Megakaryocyte stimulating factors

ABSTRACT

Novel polypeptides of human megakaryocyte stimulating factors (MSFs). Pharmaceutical compositions containing same, and methods for their preparation and use are provided.

PRIOR RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/124,557, filed on Apr. 16, 2002 now U.S. Pat. No. 7,030,223, which isa continuation of U.S. patent application Ser. No. 07/757,022, filedSep. 10, 1991, now U.S. Pat. No. 6,433,142, which is acontinuation-in-part of U.S. patent application Ser. No. 07/643,502,filed Jan. 18, 1991 now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 07/546,114, filed Jun. 29, 1990, nowU.S. Pat. No. 5,326,558, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/457,196, filed Dec. 28, 1989 now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.07/390,901, filed Aug. 8, 1989 now abandoned; these applications areeach incorporated by reference herein in their entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention relates generally to a family of novel proteinfactors which share homologous sequences and biological activities withmegakaryocyte colony-stimulating factor (meg-CSF) and which participatein the differentiation or maturation of megakaryocyte progenitors. Alsoprovided are processes for obtaining and producing the factors fromnatural sources or by artificial means, e.g., chemical synthesis orrecombinant genetic engineering techniques.

BACKGROUND OF THE INVENTION

Megakaryocytes are the hematopoietic cells, largely found in the bonemarrow, but also in peripheral blood and perhaps other tissues as well,which produce platelets (also known as thrombocytes) and subsequentlyrelease them into circulation. Megakaryocytes, like all of thehematopoietic cells of the human hematopoietic system, ultimately arederived from a primitive stem cell after passing through a complexpathway comprising many cellular divisions and considerabledifferentiation and maturation.

The platelets derived from these megakaryocytic cells are critical formaintaining hemostasis and for initiating blood clot formation at sitesof injury. Platelets also release growth factors at the site of clotformation that speed the process of wound healing and may serve otherfunctions. However, in patients suffering from depressed levels ofplatelets (thrombocytopenia) the inability to form clots is the mostimmediate and serious consequence, a potentially fatal complication ofmany therapies for cancer. Such cancer patients are generally treatedfor this problem with platelet transfusions. Other patients frequentlyrequiring platelet transfusions are those undergoing bone marrowtransplantation or patients with aplastic anemia.

Platelets for such procedures are obtained by plateletphoresis fromnormal donors. Like most human blood products, platelets for transfusionhave a relatively short shelf-life and also expose the patients toconsiderable risk of exposure to dangerous viruses, such as the humanimmunodeficiency virus (HIV) or a hepatitis virus.

Clearly the ability to stimulate endogenous platelet formation inthrombocytopenic patients with a concomitant reduction in theirdependence on platelet transfusion would be of great benefit. Inaddition, the ability to correct or prevent thrombocytopenia in patientsundergoing radiation therapy or chemotherapy for cancer would make suchtreatments safer and possibly permit increases in the intensity of thetherapy thereby yielding greater anti-cancer effects.

For these reasons considerable research has been devoted to theidentification and purification of factors involved in the regulation ofmegakaryocyte and platelet production. Although there is considerablecontroversy on this subject, the factors regulating the growth anddifferentiation of hematopoietic cells into mature megakaryocytes andthe subsequent production of platelets by these cells are believed tofall into two classes: (1) megakaryocyte colony-stimulating factors(meg-CSFs) which support the proliferation and differentiation ofmegakaryocytic progenitors in culture, and (2) thrombopoietic (TPO)factors which support the differentiation and maturation ofmegakaryocytes in vivo, resulting in the production and release ofplatelets. [See, e.g., E. Mazur, Exp. Hematol., 15:340-350 (1987).]

Each class of factors can be defined by bioassay. Factors with meg-CSFactivity support megakaryocyte colony formation, while factors with TPOactivity elicit an elevation in the numbers of circulating plateletswhen administered to animals. It is not clear how many species offactors exist that have either one or both of these activities. Forexample, human IL-3 supports human megakaryocyte colony formation and,at least in monkeys, frequently elicits an elevation in platelet count.However, IL-3 influences hematopoietic cell development in all of thehematopoietic lineages and can be distinguished from specific regulatorsof megakaryocytopoiesis and platelet formation which interactselectively with cells of the megakaryocytic lineage.

Many different reports in the literature describe factors which interactwith cells of the megakaryocytic lineage. Several putative meg-CSFcompositions have been derived from serum [See, e.g., R. Hoffman et al,J. Clin. Invest., 75:1174-1182 (1985); J. E. Straneva et al, Exp.Hematol., 15:657-663 (1987); E. Mazur et al, Exp. Hematol., 13:1164-1172(1985]. A large number of reports of a TPO factor are in the art. [See,e.g., T. P. McDonald, Exp. Hematol., 16:201-205 (1988); T. P. McDonaldet al, Biochem. Med. Metab. Biol., 37:335-343 (1987); T. Tayrien et al,J. Biol. Chem., 262: 3262-3268 (1987) and others]. From the studiesreported to date, it is not clear whether factors having activitiesidentified as meg-CSF also have TPO activity or vice versa.

Although there have been numerous additional reports tentativelyidentifying these regulatory factors, the biochemical and biologicalidentification and characterization of meg-CSF and TPO factors have beenhampered by the small quantities of the naturally occurring factorswhich are present in natural sources, e.g., blood and urine.

The present inventors have previously identified a purified meg-CSFfactor from urine described in PCT application No. WO91/02001, publishedFeb. 21, 1991, and further described in pending U.S. patent applicationSer. No. 07/643,502. This homogeneous meg-CSF, purified from urine andwhich may be produced via recombinant or synthetic techniques, ischaracterized by a specific activity in the murine fibrin clot assay ofgreater than 5×10⁷ dilution units per mg and preferably, 2×10⁸ dilutionunits per mg protein. This meg-CSF was processed from a precursorprotein encoded by an approximately 18.2 kb genomic clone which containsten exons. Two additional exons were subsequently identified outsidethis 18.2 kb segment. cDNAs made from the full length cDNA and frompartial cDNAs have been expressed in COS-1 cells and CHO cells. Thesereferences are incorporated herein by reference for the disclosure ofthat meg-CSF molecule.

There remains a need in the art for additional proteins either isolatedfrom association with other proteins or substances from their naturalsources or otherwise produced in homogeneous form, which are capable ofstimulating or enhancing the production of platelets in vivo, to replacepresently employed platelet transfusions and to stimulate the productionof other cells of the lymphohematopoietic system.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a family of humanmegakaryocyte stimulating factors (hereafter called MSFs) orbiologically active peptide fragments thereof that are substantiallyfree from association with proteins or substances with which they occurin nature. The MSF family includes non-naturally occurring MSF proteinswhich are characterized by sharing similar structure, DNA and amino acidsequences, and/or biological activity with naturally-occurring MSFs.Naturally-occurring MSFs may be isolated and purified from naturalsources. Non-naturally occurring MSFs may be prepared by chemicalsynthesis and/or recombinant genetic engineering techniques.

These MSF proteins comprise DNA sequences encoding one or more of theexon sequences described below in FIG. 1 or biologically activefragments thereof. Each MSF protein of this invention is alsocharacterized by one or a combination of the physical, biochemical,pharmacological or biological activities described herein.

Another aspect of the present invention includes a mixture of differentMSFs, and active fragments thereof.

Another aspect of the present invention includes DNA sequences thatencode the expression of these naturally occurring and non-naturallyoccurring MSFs.

Still another aspect of the present invention are recombinant DNAmolecules comprising vector DNAs and DNA sequences encoding the MSFs ofthis invention. Each DNA molecule provides the MSF DNA in operativeassociation with a regulatory sequence capable of directing thereplication and expression of each MSF in a selected host cell. Hostcells transformed with such DNA molecules for use in expressingrecombinant MSF proteins are also provided by the present invention.

The DNA molecules and transformed cells of the invention may be employedin a novel process for producing recombinant MSFs, or biologicallyactive peptide fragments thereof. A cell line, transformed with a DNAsequence encoding expression of individual MSFs or fragments thereof (ora recombinant DNA molecule as described above) in operative associationwith a suitable regulatory or expression control sequence capable ofcontrolling expression of the protein, is cultured under appropriateconditions permitting expression of the recombinant DNA. The expressedMSF protein is then harvested from the host cell, cell lysate or culturemedium by suitable conventional means.

Still a further aspect of the present invention is a process forisolating and purifying an MSF composition of the present invention or afragment thereof, or a mixture of MSFs and fragments, from naturalsources, e.g. urine. or peripheral blood leukocytes. Alternatively, theMSFs may be isolated and purified from conditioned medium or cell lysateof cells expressing recombinant MSF protein.

Another aspect of this invention provides pharmaceutical compositionscontaining a therapeutically effective amount of one or more MSFs of thepresent invention. These pharmaceutical compositions may be employed inmethods for treating disease states or disorders, for example, diseasescharacterized by a deficiency of platelets and other disorders referredto herein.

A further aspect of the invention, therefore, is a method for treatingsuch disease states by administering to a patient a therapeuticallyeffective amount of at least one MSF in a suitable pharmaceuticalcarrier. Alternatively, several MSFs may be administered in combination.These therapeutic methods may include administering in combination,simultaneously, or sequentially with an MSF, an effective amount of atleast one other TPO-like factor, meg-CSF or other cytokine, e.g, IL-3 orsteel factor, hematopoietin, interleukin, growth factor, or antibody.

Still another aspect of the present invention provides antibodiesdirected against an MSF of this invention. As part of this aspect,therefore, the invention includes cell lines capable of secreting suchantibodies and methods for their production and use in diagnostic ortherapeutic procedures.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1H present a cDNA sequence [SEQ ID NO: 1] encoding the MSFprecursor [SEQ ID NO: 2] containing sequences found in human urinarymeg-CSF, as well as sequences of other natural and artificial MSFsdisclosed herein. Each of the twelve exons has been identified byalternating solid or dashed lines extending from above the firstnucleotide in the DNA sequence encoded by that specific exon [SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25]. The correspondingamino acid sequences appear below each codon [SEQ ID NOS: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24 and 26].

FIG. 2 is a bar graph illustrating the genomic organization of the MSFgene with reference to the number of amino acids encoded by each exon.

FIGS. 3A and 3B present the modified nucleic acid sequence [SEQ ID NO:27] of MSF-K130 which was used to produce the MSF as a fusion proteinwith thioredoxin in E. coli [SEQ ID NO: 28], as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The novel family of human megakaryocyte stimulating factors (MSFs)provided by the present invention are protein or proteinaceouscompositions substantially free of association with other humanproteinaceous materials, contaminants or other substances with which thefactors occur in nature. An MSF may be purified from natural sources asa homogeneous protein, or from a selected cell line secreting orexpressing it. Mixtures of naturally occurring MSFs may be obtained fromnatural sources, or from selected cell lines by similar purificationtechniques. Another class of MSFs are “recombinant or geneticallyengineered proteins” which are defined herein as naturally occurring andnon-naturally occurring proteins prepared by chemical synthesis and/orrecombinant genetic engineering techniques, and/or a combination of bothtechniques. These MSFs may also be provided in optional association withamino acid residues and other substances with which they occur by virtueof expression of the factors in various expression systems. Recombinantor genetically-engineered MSFs of this invention may further be definedas including a polynucleotide of genomic, cDNA, semisynthetic, orsynthetic origin with sequences from the meg-CSF DNA of FIGS. 1A to 1Hwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

The MSFs of the present invention include active fragments andalternatively spliced sequences derived from the DNA [SEQ ID NO: 1] andamino acid [SEQ ID NO: 2] sequences reported in FIGS. 1A to 1H below.FIGS. 1A to 1H present a cDNA sequence [SEQ ID NO: 1] of the precursorprotein encoding urinary meg-CSF, as disclosed in published PCT patentapplication No. WO91/02001, and in U.S. patent application Ser. No.07/643,502, which are incorporated by reference herein. The nucleotidesequences [SEQ ID NO: 1] and corresponding translated amino acids [SEQID NO: 2] of FIGS. 1A to 1H are continuous in the largest identifiedcDNA [SEQ ID NO: 1] encoding the largest MSF protein [SEQ ID NO: 2], asindicated in the bar graph of FIG. 2. However in FIGS. 1A to 1H, eachexon [SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26] hasbeen identified above the DNA sequence [SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23 and 25, respectively] encoding that specific exon.While the sequence of FIGS. 1A to 1H [SEQ ID NO: 1] is believed to besubstantially complete, there may be additional, presently unidentified,exons occurring between Exons VI [SEQ ID NO: 14] and IX [SEQ ID NO: 20]or following Exon XII [SEQ ID NO: 26], which provide sequence for othermembers of the MSF family.

The exons of the meg-CSF gene [SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24 and 26] were identified by analysis of cDNA clones from COScells transfected with the gene or pieces of the gene or from cDNAsisolated from stimulated human peripheral blood lymphocytes. The firstexon [SEQ ID NO: 4], containing the initiating methionine, encodes aclassical mammalian protein secretion signal sequence. Exons II [SEQ IDNO: 6] through IV [SEQ ID NO: 8] contain the amino acid sequences of theoriginal mature urinary meg-CSF protein which most likely terminates ina region between amino acid residues 134 and 205 of FIGS. 1A to 1B,based on amino acid sequence data from the native protein. Moreprecisely, the mature human urinary meg-CSF protein terminates in theregion between amino acid residues 134 and 147. Native meg-CSF is mostlikely generated by proteolytic cleavage (endolytic cleavage followed byendolytic and/or exolytic cleavage) at or near this site in largerprecursor molecules containing additional amino acid sequences derivedfrom one or more of Exons V [SEQ ID NO: 12] through XII [SEQ ID NO: 26].

Analysis of the nucleotide and amino acid sequences of all of the exonsof the meg-CSF precursor gene [SEQ ID NOS: 3-26] have revealed someinteresting relationships to other genes. Both Exons II [SEQ ID NO: 5and 6] and III [SEQ ID NO: 7 and 8], which are related to each other,share significant sequence similarity with the somatomedin B domain ofvitronectin and of PC-1 (a surface membrane glycoprotein found on plasmacells). The Cys-rich regions of Exons II [SEQ ID NO: 5 and 6] and III[SEQ ID NO: 7 and 8] are also related to similar sequences in placentalprotein 11, a putative serine protease. A sequence containingapproximately 75 repeats of the sequence Lys-Pro-Thr which is present inExon VI [SEQ ID NO: 13 and 14], resembles similar repeat sequences whichhave been found in membrane-bound glycoproteins and precursor proteins,such as spasmolysin.

Exons VI [SEQ ID NO: 13 and 14] through XII [SEQ ID NO: 25 and 26], inparticular Exons VII [SEQ I) NO: 15 and 16], VIII [SEQ ID NO: 17 and 18]and IX [SEQ ID NO: 19 and 20], also contain sequences which are found invitronectin distinct from the somatomedin B domain. These exons encode asequence found in vitronectin and members of the collagenase family,e.g., human stromelysin. Another functional domain of vitronectinincluding the RGD adhesion sequence known to bind integrins is not foundin any of the exons of the meg-CSF gene. The functions of the amino acidsequences from Exons V [SEQ ID NO: 11 and 12] through XII [SEQ ID NO: 25and 26] have not yet been determined, but may play roles which effectthe three dimensional structure, i.e., folding of the molecule.

During the course of the analysis of the structure of the 18.2 kb“meg-CSF” gene, it was discovered that the primary RNA transcript isspliced in a variety of ways to yield a family of mRNAs each encodingdifferent MSF proteins. In addition, these precursor proteins can beprocessed in different ways to yield different mature MSF proteins.Thus, a family of MSF's exist in nature, including the meg-CSF which wasisolated from urine from the bone marrow transplant patients. Allmembers of this family are believed to be derived from the 18.2 kbmeg-CSF gene plus a few additional exons, found in the peripheral bloodleukocyte cDNA located just downstream from the 3′ end of the 18.2 kbgene.

The entire 18.2 kb genomic sequence inserted as a NotI fragment inbacteriophage lambda DNA was deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, USA underaccession # ATCC 40856. The 3′ PBL partial cDNA clone beginning onethird of the way from the 5′ end of Exon VI [SEQ ID NOS: 13 and 14](containing the SnaB site) extending through Exon XII [SEQ ID NOS: 25and 26] is stored at Genetics Institute, Inc. and can be readily madeavailable without restrictions by deposit at the ATCC upon an indicationof allowance of this application.

This invention also contemplates the construction of “recombinant orgenetically-engineered” classes of MSFs, which may be generated usingdifferent combinations of the amino acid sequences [SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24 and 26] of the exons of FIGS. 1A to 1G.Some of these novel MSFs may be easier to express and may have differentbiological properties than the native urinary meg-CSF.

Without being bound by theory, and based on analysis of the naturallyoccurring meg-CSF sequence of FIGS. 1A to 1H [SEQ ID NO: 1 and 2], it isspeculated that Exon I [SEQ ID NO: 3 and 4] is necessary for properinitiation and secretion of the MSF protein in mammalian cells; and thatExon XII [SEQ ID NO: 25 and 26] is necessary for termination of thetranslation of the naturally occurring protein. Exons II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] arebelieved to contain the sequences essential to biological activity ofthe MSF. Exons V [SEQ ID NO: 11 and 12] and VI [SEQ ID NO: 13 and 14]may be related to activity of the factor, but are also implicated in thestability and binding of the molecule. Alternately spliced forms of MSFcDNAs containing either Exon V [SEQ ID NO: 11 and 12] or VI [SEQ ID NO:13 and 14] have been observed. Exon V [SEQ ID NO: 11 and 12] and Exon VI[SEQ ID NO: 13 and 14] are also believed to play a role in the synergyof MSF with other cytokines. No alternative splicing has yet beenobserved between Exons VI [SEQ ID NO: 13 and 14] and XII [SEQ ID NO: 25and 26]. However, such splicing in the region of Exons VI [SEQ ID NO: 13and 14] through XI [SEQ ID NO: 25 and 26] may occur. Exons V [SEQ ID NO:11 and 12] through XII [SEQ ID NO: 25 and 26] are believed to beimplicated in the processing or folding of the appropriate structure ofthe resulting factor. For example, one or more of Exons V [SEQ ID NO: 11and 12] through XII [SEQ ID NO: 25 and 26] may contain sequences whichdirect proteolytic cleavage, adhesion, or extracellular matrixprocessing.

Both naturally occurring MSFs and non-naturally-occurring MSFs may becharacterized by various combinations of alternatively spliced exons ofFIGS. 1A to 1H, with the exons spliced together in differing orders toform different members of the MSF family. At a minimum at least one ofthe group consisting of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7and 8] and IV [SEQ ID NO: 9 and 10] and a biologically active fragmentthereof is present in a MSF.

Naturally-occurring MSFs may possess at least Exon I [SEQ ID NO: 3 and4], which contains both an initiating methionine necessary fortranslation and a secretory leader for secretion of the factor frommammalian cells, and one or more additional exons of FIGS. 1A to 1H. Ofthese additional exons, at least one is selected from the groupconsisting of Exons II [SEQ ID NO: 5 and 6], m [SEQ ID NO: 7 and 8] andIV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof. Anexemplary MSF [SEQ ID NO: 55 and 56] of this class includes a proteinrepresented by the spliced-together arrangement of Exons I [SEQ ID NO: 3and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8]. Still anotherexemplary MSF [SEQ ID NO: 57 and 58] of this class includes Exons I [SEQID NO: 3 and 4], III [SEQ ID NO: 5 and 6], V [SEQ ID NO: 11 and 12] andVI [SEQ ID NO: 13 and 14].

Other naturally occurring MSFs may possess both Exon I [SEQ ID NO: 3 and4] and Exon XII [SEQ ID NO: 25 and 26], which latter exon contains atermination codon for translation, and at least one additional exonselected from Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8]and IV [SEQ ID NO: 9 and 10], and a biologically active fragmentthereof. It is speculated that both the initiating Met of Exon I [SEQ IDNO: 3 and 4] and the termination codon of Exon XII [SEQ ID NO: 25 and26] are required to produce an active, properly folded,naturally-occurring MSF in a eukaryotic cell. Thus naturally-occurringMSFs may contain at least Exons I [SEQ ID NO: 3 and 4] and XII [SEQ IDNO: 25 and 26] and another exon. An exemplary MSF [SEQ ID NO: 61] ofthis class includes a protein [SEQ ID NO: 62] represented by thespliced-together arrangement of exons selected from Exons I through XIIof FIGS. 1A to 1G [SEQ ID NO: 3-26]. Still another exemplary MSF of thisclass [SEQ ID NO: 29] includes a protein [SEQ ID NO: 30] encoded by thespliced Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] andXII [SEQ ID NO: 25 and 26]. Another MSF of this class [SEQ ID NO: 31 and32] is formed by spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10] andXII [SEQ ID NO: 25 and 26]. Still another MSF [SEQ ID NO: 33 and 34] ofthis class includes the spliced together sequences of Exons I [SEQ IDNO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and XII[SEQ ID NO: 25 and 26]. Another MSF sequence [SEQ ID NO: 35 and 36] isformed by spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO:7 and 8] and XII [SEQ ID NO: 25 and 26]. Yet a further example of an MSFof this class [SEQ ID NO: 37 and 38] is formed by the spliced togetherarrangement of Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8],IV [SEQ ID NO: 9 and 10] and XII [SEQ ID NO: 25 and 26].

Another class of naturally occurring MSFs may be characterized by thepresence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10],or a biologically active fragment thereof, and all of Exons VI throughXII [SEQ ID NOS: 13-26]. An exemplary MSF [SEQ ID NO: 39 and 40] of thisclass includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ IDNO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], and VIthrough XII [SEQ ID NO: 13-26]. Another MSF of this class [SEQ ID NO: 41and 42] is formed by spliced together Exons I [SEQ ID NO: 3 and 4], II[SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], and VI through XII [SEQID NO: 13-26]. Still another MSF sequence [SEQ ID NO: 43 and 44] isformed from spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ IDNO: 7 and 8], and VI through XII [SEQ ID NO: 13-26]. Another MSFsequence [SEQ ID NO: 45 and 46] of this class includes spliced togetherExons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], IV and VIthrough XII [SEQ ID NO: 13-26].

Still another class of naturally occurring MSFs may be characterized bythe presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II[SEQ ID NO: 5 and 6]; III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and10], and a biologically active fragment thereof; and Exons V through XII[SEQ ID NO: 11-26]. An exemplary MSF [SEQ ID NO: 47 and 48] of thisclass includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ IDNO: 5 and 6], III [SEQ ID NO: 7 and 8], and V through XII [SEQ ID NO:11-26]. Another MSF [SEQ ID NO: 141 and 142] of this class is formed byspliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8],and V through XII [SEQ ID NO: 11-26]. Still another MSF [SEQ ID NO: 49and 50] sequence is formed from spliced together Exons I [SEQ ID NO: 3and 4], II [SEQ ID NO: 5 and 6], and V through XII [SEQ ID NO: 11-26].Another MSF sequence [SEQ ID NO: 51 and 52] of this class includesspliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8],IV [SEQ ID NO: 9 and 10] and V through XII [SEQ ID NO: 11-26].

Another class of naturally occurring MSFs may be characterized by thepresence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10],and a biologically active fragment thereof; Exon V [SEQ ID NO: 11 and12], and Exons VII through XII [SEQ ID NO: 15-26]. An exemplary MSF [SEQID NO: 53 and 54] of this class includes spliced together Exons I [SEQID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV[SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VII through XII [SEQID NO: 15-26]. Another MSF of this class [SEQ ID NO: 63 and 64] isformed by Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], V [SEQID NO: 11 and 12] and VII through XII [SEQ ID NO: 15-26] in a splicedtogether form. Still another MSF sequence [SEQ ID NO: 65 and 66] isformed from spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ IDNO: 5 and 6], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VIIthrough XII [SEQ ID NO: 15-26]. Another MSF sequence [SEQ ID NO: 67 and68] of this class includes Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO:7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VIIthrough XII [SEQ ID NO: 15-26] spliced together.

Yet another class of naturally occurring MSFs may be characterized bythe presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II[SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and10] and a biologically active fragment thereof, at least one of Exons Vthrough XI [SEQ ID NO: 11-24]; and Exon XII [SEQ ID NO: 25 and 26]. Anexemplary MSF [SEQ ID NO: 69 and 70] of this class includes splicedtogether Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], X[SEQ ID NO: 21 and 22] and XII [SEQ ID NO: 25 and 26]. Another MSF [SEQID NO: 71 and 72] of this class is formed by spliced together Exons I[SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8],VIII [SEQ ID NO: 17 and 18], IX [SEQ ID NO: 19 and 20] and XIII [SEQ IDNO: 25 and 26]. Still another MSF sequence [SEQ ID NO: 73 and 74] isformed from spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ IDNO: 7 and 8], VI [SEQ ID NO: 13 and 14] and XII [SEQ ID NO: 25 and 26].Another MSF sequence [SEQ ID NO: 75 and 76] of this class includesspliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID. NO: 5 and 6],IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], VII [SEQ ID NO: 15and 16] and XII [SEQ ID NO: 25 and 26].

For recombinant or genetically engineered MSFs, Exon I [SEQ ID NO: 3 and4] may be replaced by a synthetic or heterologous sequence containing aninitiating Met and a selected secretory leader designed for use in aselected expression system (hereafter referred to for simplicity as“artificial Exon I”). The natural Exon I [SEQ ID NO: 3 and 4] may becompletely absent for intracellular expression in a bacterial host, suchas E. coli. A secretory leader may be selected from among knownsequences for secretion of proteins from a variety of host cells. Anumber of secretory leaders are known for bacterial cells, yeast cells,mammalian cells, insect cells and fungi which may be useful as hostcells for expression of a recombinant or genetically-engineered MSF. Theconstruction of an appropriate genetically engineered Exon I sequencecontaining a secretory leader and initiating Met is within the skill ofthe art with resort to known sequences and techniques. Thus, one classof recombinant MSFs may be characterized by a genetically-engineeredExon I in place of the naturally occurring Exon I [SEQ ID NO: 3 and 4]of FIG. 1A.

Additionally, the termination codon supplied by Exon XII [SEQ ID NO: 25and 26] to naturally occurring MSFs may be replaced by inserting into,or after, a selected exon of FIGS. 1A to 1H a termination codon suitableto a selected host cell (hereafter referred to for simplicity as“artificial termination codon”). The construction of an appropriate MSFsequence containing a termination codon is within the skill of the artwith resort to known codons for a variety of host cells and conventionaltechniques. Thus one class of recombinant MSFs may be characterized bythe presence of an artificial termination codon.

One class of recombinant MSFs include a naturally-occurring Exon I [SEQID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically activefragment thereof; and an artificial termination codon. An example ofsuch an MSF is MSF-K130 [SEQ ID NO: 77 and 78] and MSF-N141 [SEQ ID NO:79 and 80], among others described in detail below.

Another class of recombinant MSFs include an artificial Exon I, at leastone of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV[SEQ ID NO: 9 and 10], and a biologically active fragment thereof; andExon XII [SEQ ID NO: 25 and 26].

Still another class of recombinant MSFs include an artificial Exon I, atleast one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] andIV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof;and an artificial termination codon.

Another class of recombinant, genetically-engineered MSFs includegenetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and abiologically active fragment thereof; and all of Exons V through XII[SEQ ID NO: 11-26].

Still another class of recombinant MSFs may be characterized by thepresence of genetically-engineered Exon I, at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10],and a biologically active fragment thereof; and Exons VI through XII[SEQ ID NO: 13-26].

Another class of recombinant MSFs may be characterized by the presenceof genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and abiologically active fragment thereof; Exon V [SEQ ID NO: 11 and 12], andExons VII through XII [SEQ ID NO: 15-26].

Yet another class of recombinant MSFs may be characterized by thepresence of genetically-engineered Exon I, at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10]and a biologically active fragment thereof; at least one of Exons Vthrough XI [SEQ ID NO: 11-24]; and an artificial termination codon.

Another class of recombinant MSFs is characterized bygenetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and abiologically active fragment thereof; all of Exons V through XI [SEQ IDNO: 11-24], with an artificial termination codon either inserted into,or added onto a selected last exon of the sequence.

Another class of recombinant MSFs is characterized bygenetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and abiologically active fragment thereof; all of Exons VI through XI [SEQ IDNO: 13-24], with an artificial termination codon.

Another class of recombinant MSFs may be characterized by the presenceof native Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ IDNO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], anda biologically active fragment thereof, and all of Exons V through XI[SEQ ID NO: 11-24], with an artificial termination codon.

Still another class of recombinant MSFs may be characterized by thepresence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10],and a biologically active fragment thereof; and all of Exons VI throughXI [SEQ ID NO: 13-24], With an artificial termination codon.

Yet another class of recombinant MSFs may be characterized by thepresence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10]and a biologically active fragment thereof; at least one of Exons Vthrough XI [SEQ ID NO: 11-24]; and an artificial termination codon.

A further class of recombinant, genetically-engineered MSFs ischaracterized by the complete absence of an Exon I. Such MSFs are usefulfor intracellular expression in bacterial cells, such as E. coli. TheseMSFs may comprise at least one of Exons II [SEQ ID NO: 5 and 6], III[SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] and a biologicallyactive fragment thereof; optionally one or more exons from Exons Vthrough XII [SEQ ID NO: 11-26]. In the absence of Exon XII [SEQ ID NO:25 and 26], an artificial termination codon may be inserted into orafter the last preferred carboxy terminal exon. Exemplary MSFs of thisinvention are MSF-234 [SEQ ID NO: 81 and 82] and MSF 236 [SEQ ID NO: 83and 84] described below in detail.

In another class of naturally-occurring or non-naturally occurring MSFs,either the sequences of Exon VIII [SEQ ID NO: 17 and 18] and Exon IX[SEQ ID NO: 19 and 20] will be present together, or neither of these twoexons will be present. This is primarily due to frame shifts betweenthese exons and the remaining MSF exons.

Finally an MSF [SEQ ID NO: 61 and 62] exists which contains all twelveexons [SEQ ID NO: 3-26].

While the above described MSF sequence structures will provide forprecursor MSFs capable of being processed naturally, or by a host cellexpression system, into mature MSF proteins, it is considered thatmature, processed form of the proteins produced in eukaryotic systemswill be missing all or part of Exon I [SEQ ID NO: 3 and 4]. Perhaps themature proteins may be missing a portion of Exon II [SEQ ID NO: 5 and 6]as well, in order to remove the leader sequence from the processed form.The processed forms of MSF proteins may also be missing substantialsequences from the carboxy terminus. For example, sequences from Exons Vthrough XII [SEQ ID NO: 11-26] may be absent in mature, processed MSFproteins. As another example, sequences from Exons VI through XII [SEQID NO: 13-26] may be absent in mature, processed MSF proteins. As stillanother example, sequences from Exons VII through XII [SEQ ID NO: 15-26]may be absent in mature, processed MSF proteins. In such manner humanurinary meg-CSF, an illustrative naturally-occurring MSF [SEQ ID NO: 81and 82], has a mature protein sequence characterized by the presence ofExons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ IDNO: 9 and 10] in a predominantly homodimeric form.

Selected examples of artificial MSFs were prepared by the followingmethods. During the analysis of the meg-CSF gene, a contiguous cDNA wasconstructed containing Exons I through VI [SEQ ID NO: 3-14], in whichthe primary translation product was artificially interrupted byinserting artificial termination codons at different positions in ExonsIV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], and VI [SEQ ID NO:13 and 14] near the point at which the original meg-CSF was believed tobe processed, i.e. the region between amino acid residues 134 and 209.These cDNAs were transfected into COS cells and the resultingsupernatants were tested for meg-CSF activity. Through this process,several different biologically active MSFs were identified.

One MSF of the present invention is characterized by the DNA sequence[SEQ ID NO: 77] extending from nucleotide number 1 of Exon I throughnucleotide number 390 of Exon IV, encoding an amino acid sequence [SEQID NO: 78] which is a continuous fusion in frame extending from aminoacid I of Exon I [SEQ ID NO: 3 and 4] through amino acid 130 of Exon IV[SEQ ID NO: 9 and 10] of the sequence of FIG. 1A, with a terminationcodon inserted thereafter. The predicted molecular weight of this MSF[SEQ ID NO: 77 and 78] is approximately 11.6 kD. In 4-20% gradientsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),under reducing conditions, a major species of molecular weight ofapproximately 19 kD has been detected. This MSF [SEQ ID NO: 77 and 78]does not bind heparin under the standard binding conditions of 20 mMtris and pH 7.4. The construction and expression of this molecule,called MSF-K130 [SEQ ID NO: 77 and 78], is described in detail inExample 3.

Under SDS-PAGE non-reducing conditions, the molecular weight ranged fromabout 20 to about 50 kD. Upon expression in COS-1 cells, this MSF cDNAsequence [SEQ ID NO: 77] produces a mixture of monomeric andhomo-dimeric species. The homodimer has exhibited activity in the fibrinclot assay of Example 8. In the inventors' hands, this is one of themost easily expressed MSF [SEQ ID NO: 77 and 78] which is also highlyactive in the fibrin clot assay. The MSF expressed by this sequence [SEQID NO: 77 and 78] in mammalian cells approximates the structure andproperties of the native human urinary meg-CSF.

Another MSF of the present invention, called MSF-N141 [SEQ ID NO: 79 and80], is characterized by a nucleotide sequence [SEQ ID NO: 79] extendingfrom nucleotide number 1 of Exon I [SEQ ID NO: 3] through nucleotidenumber 423 of Exon IV [SEQ ID NO: 9], encoding an amino acid sequence[SEQ ID NO: 80] extending from amino acid 1 through amino acid 141 ofthe sequence of FIG. 1A with an artificial termination codon insertedthereafter. The predicted molecular weight of this MSF [SEQ ID NO: 79and 80] is approximately 13.2 kD. In 4-20% SDS-PAGE under reducingconditions, a major species of molecular weight of approximately 21 kDhas been detected. This MSF [SEQ ID NO: 79 and 80] binds heparin understandard binding conditions. Upon expression in COS-1 cells, this MSFcDNA sequence [SEQ ID NO: 79] produces a mixture of monomeric andhomo-dimeric species. The monomeric form is the major form secreted byCOS-1 cells. The COS cell conditioned medium from this transfection alsoyielded biological activity in the murine fibrin clot assay. Thehomodimeric form is the major species secreted by CHO cells.

Still another MSF of the present invention, MSF-S172 [SEQ ID NO: 87 and88], is characterized by a nucleotide sequence [SEQ ID NO: 87] extendingfrom nucleotide numbers 1 of Exon I through 516 of Exon V, encoding anamino acid sequence [SEQ ID NO: 88] extending from amino acid 1 throughamino acid 172 of the sequence of FIG. 1A with an artificial terminationcodon inserted thereafter. The predicted molecular weight of this MSF[SEQ ID NO: 87 and 88] is approximately 16.2 kD, and in 4-20% SDS-PAGEunder reducing conditions, a major species of molecular weight ofapproximately 23.5 kD has been detected. This MSF [SEQ ID NO: 87 and 88]also binds to heparin under standard binding conditions.

A further MSF of the present invention, MSF-R192 [SEQ ID NO: 89 and 90],is characterized by a nucleotide sequence [SEQ ID NO.: 89] extendingfrom nucleotide number 1 of Exon I through 576 of Exon V, encoding anamino acid sequence [SEQ ID NO: 90] extending from amino acid 1 throughamino acid 192 of the sequence of FIG. 1A with an artificial terminationcodon inserted thereafter. The predicted molecular weight of this MSF[SEQ ID NO: 89 and 90] is approximately 18.4 kD, and in 4-20% SDS-PAGEunder reducing conditions, a major species of molecular weight ofapproximately 27 kD has been detected. This MSF [SEQ ID NO: 89 and 90]also binds to heparin under standard conditions.

Yet another MSF of the present invention, called MSF-K204 [SEQ ID NO: 91and 92], is characterized by a nucleotide sequence [SEQ ID NO: 91]extending from nucleotide numbers 1 of Exon I through 612 of Exon VI,encoding an amino acid sequence [SEQ ID NO: 92] extending from aminoacid 1 through amino acid 204 of the sequence of FIGS. 1A to 1B. Thepredicted molecular weight of this MSF [SEQ ID NO: 91 and 92] isapproximately 19.8 kD. In 4-20% SDS-PAGE under reducing conditions, amajor species of molecular weight of approximately 28 kD has beendetected. This MSF [SEQ ID NO: 91 and 92] also binds to heparin understandard conditions.

Still a further MSF of the present invention, called MSF-K209 [SEQ IDNO: 93 and 94], is characterized by a nucleotide sequence [SEQ ID NO:93] extending from nucleotide numbers 1 of Exon I through 627 of ExonVI, encoding an amino acid sequence [SEQ ID NO: 94] extending from aminoacid 1 through amino acid 209 of the sequence of FIGS. 1A to 1B with anartificial termination codon inserted thereafter. The predictedmolecular weight of this MSF [SEQ ID NO: 93 and 94] is approximately20.4 kD, and in 4-20% SDS-PAGE under reducing conditions, a majorspecies of molecular weight of approximately 29 kD has been detected.This MSF [SEQ ID NO: 93 and 94] also binds to heparin under standardconditions.

Another MSF of the present invention, MSF-D220 [SEQ ID NO: 95 and 96],is characterized by a nucleotide sequence [SEQ ID NO: 95] extending fromnucleotide numbers 1 of Exon I through 660 of Exon VI, encoding an aminoacid sequence [SEQ ID NO: 96] extending from amino acid 1 through aminoacid 220 of the sequence of FIGS. 1A to 1B with an artificialtermination codon inserted thereafter. The predicted molecular weight ofthis MSF [SEQ ID NO: 95 and 96] is approximately 21.6 kD, and in 4-20%SDS-PAGE under reducing conditions, a major species of molecular weightof approximately 30 kD has been detected. This MSF [SEQ ID NO: 95 and96] also binds to heparin under standard conditions.

Although in all of the above-described MSFs, the amino and carboxytermini of each MSF is defined precisely, it is to be understood thataddition or deletion of one or several amino acids (and consequent DNAcoding region) from either end of any of the MSFs (or from either end ofany of the exons forming the spliced MSFs) is not likely tosignificantly alter the properties of the particular MSF. Such truncatedMSFs which also retain MSF biological activities are also encompassed bythis disclosure. The deliberate insertion of artificial terminationcodons at other positions in the MSF sequences can provide other membersof the MSF family.

The alternatively spliced MSFs of the present invention arecharacterized by amino acid sequences containing at least two exons andless than twelve exons of FIGS. 1A to 1H as described above, which exonsare spliced together in various arrangements. Several representative“alternatively-spliced” naturally occurring MSF sequences have beenidentified by polymerase chain reaction (PCR) of cDNA prepared fromvarious cell lines. The sequences of these MSFs were confirmed byhybridization to oligonucleotides spanning exon junctions, molecularweight of PCR fragments, and by DNA sequence in one case. A secondmethod of obtaining MSF sequences involved natural isolation of cDNAsfrom a HeLa cDNA library. The molecular weights of these MSFs werecalculated.

In the PCR technique, the primers extended across exon junctions betweenExons I through VI [SEQ ID NO: 3-14]. Primers for use between Exons VIand XII [SEQ ID NO: 13-26]; are currently being designed. Therefore,these seven exons may all be present in these MSFs. Alternatively, noexon from Exon VI through XII [SEQ ID NO: 13-26] may be present. Stillalternatively one or more of Exon VI through XII [SEQ ID NO: 13-26] maybe present in these representative alterrately spliced MSFs.

For example, the 5′ end of one such MSF, called MSF-136 [SEQ ID NO: 97and 98], identified by PCR is characterized by a contiguous amino acidsequence containing amino acid 1 to 25 of Exon I [SEQ ID NO: 4](nucleotides 1 through 76 of FIG. 1A [SEQ ID NO: 3]) joined in frame toamino acid 67 to 106 of Exon III [SEQ ID NO: 8] (nucleotides 200 through319 [SEQ ID NO: 7]), joined in frame to amino acid 200 to about 250 ofExon VI [SEQ ID NO: 100] (nucleotides 598 through about 748 [SEQ ID NO:99]). Although not identified by a PCR primer, additional 3′ sequencemay be present in this MSF [SEQ ID NO: 97 and 98], as in each of thebelow described PCR-identified sequences. This 5′ MSF sequence [SEQ IDNO: 97 and 98] has been detected in the cDNA of the following celllines: the osteosarcoma cell line U20S (ATCC No. HTB96), the small celllung carcinoma H128 (ATCC No. HTB120), the neuroblastoma SK-N-SH (ATCCNo. HTB11), the neuroblastoma SK-N-MC (ATCC No. HTB 10), theerythroleukemia cell line OCIM1, the erythroleukemia cell line K562(ATCC No. CCL243) following culture in the presence or absence ofphorbol myristate acetate, the hepatoma cell line HEPG2 (ATCC No.HB8065) and in stimulated peripheral blood leukocytes from normalvolunteers (PBLs). The presence of this MSF-136 [SEQ ID NO: 97 and 98]indicates that a naturally-occurring alternately spliced MSF maycomprise Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], VI [SEQID NO: 13 and 14] and optionally one or more of Exons VII through XII[SEQ ID NO: 15-26]. An artificial MSF mimicking this structure may havean artificial termination codon inserted within or after Exon VI [SEQ IDNO: 13 and 14].

Another PCR-identified 5′ MSF sequence, called MSF-1236 [SEQ ID NO: 101and 102], is characterized by a contiguous amino acid sequence [SEQ IDNO: 102] containing amino acid 1 to 25 [SEQ ID NO: 4] (nucleotides 1through 76 [SEQ ID NO: 3]) of Exon I joined in frame to amino acid 26 to66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of ExonII, joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to aminoacid 200 to about 250 [SEQ ID NO: 100] (nucleotides 598 through about748 [SEQ ID NO: 99]) of Exon VI. This 5′ MSF sequence [SEQ ID NO: 101and 102] has been detected by PCR analysis of the following cell lines:U20S, H128, SK-N-SH, SK-N-MC, the neuroglioma epithelial-like cell lineH4 (ATCC No. HTB148), OCIM1, K562, K562 in the presence of PMA, theerythroleukemia cell line HEL (ATCC No. TIB180) in the presence of PMA,OCIM2, HEPG2 and stimulated PBLs. The presence of this MSF-1236 [SEQ IDNO: 101 and 102] indicates that a naturally-occurring alternatelyspliced MSF may comprise Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5and 6], III [SEQ ID NO: 7 and 8], VI [SEQ ID NO: 13 and 14] andoptionally one or more of Exons VII through XII [SEQ ID NO: 15-26]. Arecombinant MSF mimicking this structure may have an artificialtermination codon inserted within or after Exon VI [SEQ ID NO: 13 and14].

Still another MSF [SEQ ID NO: 103 and 104] according to this inventionis characterized by a contiguous amino acid sequence containing aminoacid 1 to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) ofExon I joined in frame to amino acid 26 to 66 [SEQ ID NO: 6](nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II, joined in frameto amino acid 67 to 106 [SEQ. ID NO: 8] (nucleotides 200 through 319[SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to 156[SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of Exon IV,joined in frame to amino acid 200 to 1140 [SEQ ID NO: 14] (nucleotides598 through 3421 [SEQ ID NO: 13]) of Exon VI. This MSF-12346 [SEQ ID NO:103 and 104] has been detected by PCR analysis of the following celllines: U20S, SK-N-SH, SK-N-MC, OCIM1 in the presence of PMA, K562 in thepresence of PMA, HEPG2 and stimulated PBLs. The presence of thisMSF-12346 [SEQ ID NO: 103 and 104] indicates that a naturally-occurringalternately spliced MSF may comprise Exons I [SEQ ID NO: 3 and 4], II[SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and10], VI [SEQ ID NO: 13 and 14] and optionally one ormore of Exons VIIthrough XII [SEQ ID NO: 15-26]. A recombinant MSF mimicking thisstructure may have an artificial termination codon inserted within orafter Exon VI [SEQ ID NO: 13 and 14].

Another MSF sequence of this invention may include MSF-1234 [SEQ ID. NO:105 and 106], characterized by a contiguous amino acid sequencecontaining amino acid 1 to 25 [SEQ ID NO: 4] (nucleotides 1 through 76[SEQ ID NO: 3]) of Exon I (signal peptide) joined in frame to amino acid26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) ofExon II, joined in frame to amino acid 67 to 106 [SEQ ID NO: 8](nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined inframe to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through469 [SEQ ID NO: 9]) of Exon IV. This sequence optionally has a 3′sequence comprising one or more of Exons V through XII [SEQ ID NO:11-26]. This sequence may also contain an artificial termination codoninserted within or after any selected C-terminal exon.

Still another MSF sequence, MSF-134 [SEQ ID NO: 107 and 108], ischaracterized by a contiguous amino acid sequence containing amino acidI to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) of ExonI (signal peptide) joined in frame to amino acid 67 to 106 [SEQ ID NO:8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined inframe to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through469 [SEQ ID NO: 9]) of Exon IV. This sequence [SEQ ID NO: 107 and 108]optionally has a 3′ sequence comprising one or more of Exons V throughXII [SEQ ID NO: 11-26]. This sequence [SEQ ID NO: 107 and 108] may alsocontain an artificial termination codon inserted within or after anyselected C-terminal exon.

Two examples of MSFs that may be useful for bacterial intracellularexpression include MSF-234 [SEQ ID NO: 81 and 82], characterized by acontiguous amino acid sequence containing amino acid 26 to 66 [SEQ IDNO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II joined inframe to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to156 [SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of ExonIV; and MSF-236 [SEQ ID NO: 83 and 84], characterized by a contiguousamino acid sequence containing amino acid 26 to 66 [SEQ ID NO: 6](nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II joined in frameto amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQID NO: 7]) of Exon III, joined in frame to amino acid 200 to 1140 [SEQID NO: 14] (nucleotides 598 through 3421 [SEQ ID NO: 13]) of Exon VI.These sequences [SEQ ID NOS: 81 and 82; SEQ ID NOS: 83 and 84] eachoptionally may have a 3′ sequence comprising one or more of Exons Vthrough XI [SEQ ID NO: 11-26]. These sequences [SEQ ID NOS: 81 and 82;SEQ ID NOS: 83 and 84] may also contain an artificial termination codoninserted within or after any selected C terminal exon.

It is further contemplated by the present invention that other MSFswhich may be characterized by having MSF biological activities and whichmay be useful as research agents, diagnostic agents or as therapeuticagents, include factors having other combinations and arrangements oftwo or more of the exons identified in FIGS. 1A to 1H [SEQ ID NO: 1 and2]. The splicing of the exons to form recombinant MSFs may beaccomplished by conventional genetic engineering techniques or chemicalsynthesis, as described herein.

Additionally, analogs of MSFs are included within the scope of thisinvention. An MSF analog may be a mutant or modified protein orpolypeptide that retains MSF activity and preferably has a homology ofat least about 50%, more preferably about 70%, and most preferablybetween about 90 to about 95% to human urinary meg-CSF. Still other MSFanalogs are mutants that retain MSF activity and preferably have ahomology of at least about 50%, more preferably about 80%, and mostpreferably between 90 to 95% to MSF-K130 [SEQ ID NO: 77 and 78] and theother truncated MSFs. Typically, such analogs differ by only 1, 2, 3, or4 codon changes. Examples include MSFs with minor amino acid variationsfrom the amino acid sequences of native or recombinant meg-CSF, or anyof the above-described MSFs, in particular, conservative amino acidreplacements. Conservative replacements are those that take place withina family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cystine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarconservative replacement of an amino acid with a structurally relatedamino acid will not have a major effect on the MSF activity, especiallyif the replacement does not involve an amino acid at the active site ofthe MSF.

The MSFs of this invention may form monomers or homo- or hetero-dimerswhen expressed in suitable expression systems, due to the presence ofcysteine-rich sequences in the exons. As indicated above, two specifichomodimeric forms have been identified, namely the MSF-K130 [SEQ ID NO:77 and 78] characterized by the sequence of amino acid 1 through 130 ofFIG. 1A, and the MSF-N141 [SEQ ID NO: 79 and 80] characterized by thesequence of amino acid 1 through 141 of FIG. 1A. These homodimeric formswere found as abundant forms of these proteins. However, these proteinsexisted in mixtures of other dimeric and monomeric forms.

Other MSFs of this invention are predominantly monomers rather thanmixtures, such as the MSFs characterized by the sequence of amino acids1 through 209 of FIG. 1A to 1B [SEQ ID NO: 93 and 94], or amino acids Ithrough 172 of FIG. 1A [SEQ 1D NO: 87 and 88], among others.

MSFs of the present invention may act directly on megakaryocyteprogenitor cells and/or megakaryocytes. MSFs may act indirectly onaccessory cells, such as macrophages and T cells, to produce cytokineswhich stimulate megakaryocyte colony functions. Specifically, MSFsdisplay megakaryocyte colony stimulating activity. Another MSF activityis the promotion of megakaryocyte maturation. The active MSFcompositions of the present invention have biological activity in themurine fibrin clot megakaryocyte colony formation assay. For example,the MSF characterized by the amino acid sequence amino acid 1 of Exon Ithrough amino acid 130 of Exon IV (MSF-K130) [SEQ ID NO: 77 and 78] hasa specific activity of greater than approximately 1×10⁷ dilutionunits/mg protein.

MSFs may also be used in synergy with other cytokines. For example, MSFsalso display enhancement of IL-3-dependent megakaryocyte colonyformation. MSFs may display enhancement of steel factor-dependentmegakaryocyte colony formation [See, K. M. Zsebo et al, Cell, 63:195-201(1990) for the identification of steel factor, also known as SCF. Thisdocument is incorporated by reference herein]. Together, thesecytokines, IL-3 and steel factor, have been shown to stimulate increasedmegakaryocyte colony formation in vitro. In addition, IL-3 has beenshown to elevate the level of platelets in non-human primates in vivo.

It is contemplated that all MSFs encoded by the combinations ofsequences selected from Exons I through XII [SEQ ID NO: 61 and 62] asreported above will have MSF biological activity, for example, activityin the murine fibrin clot assay, either alone or in combination withother cytokines. All modified or mutant MSF peptides or polypeptides ofthis invention, including the spliced forms of MSF, may be readilytested for activity in the megakaryocyte fibrin clot assay, either aloneor in combination with other known cytokines including IL-3, steelfactor or GM-CSF. Other cytokines which may be useful in combinationwith MSF include G-CSF, CSF-1, GM-CSF, IL-1, IL-4, erythropoietin, IL-6,TPO, M-CSF1, CSF-1₁, meg-CSF and IL-7.

These MSFs may also have biological or physiological activities inaddition to the ability to stimulate the growth and development ofcolonies consisting of intermediate and large size megakaryocytes inculture in the assay using murine bone marrow target cells. In themurine fibrin clot megakaryocyte colony formation assay, an MSFcomposition of the present invention stimulates the growth of multiplecolony types, but at least 50% of the colonies are pure megakaryocyticor mixed lineage colonies having significant numbers of megakaryocytes.The exact composition of colony types may vary with different assayconditions (fetal calf serum lots, etc). Among themegakaryocyte-containing colonies, typically 50-70% are puremegakaryocytic in composition.

In the murine agar megakaryocyte colony formation assay, an MSF of thepresent invention may stimulate colonies of megakaryocytes. In somecases, the particular MSF may not by itself stimulate megakaryocytecolony formation, but rather may enhance megakaryocyte colony formationsupported by other factors, such as IL-3 or steel factor; or it maysynergize with other factors, such as IL-11, which alone is not capableof supporting megakaryocyte colony formation in the fibrin clot assay.

It is presently anticipated that maximal biological activities of theseMSFs in vitro may be achieved by activating the factors with acid, ordenaturing conditions in SDS-PAGE, or by reverse phase high pressurechromatography (RP-HPLC). With both the native urinary protein and therecombinant MSF-K130 [SEQ ID NO: 77 and 78], an increase in the numberof units of activity has been routinely detected after SDS-PAGE andRP-HPLC.

The present invention also encompasses MSF-encoding DNA sequences, freeof association with sequences and substances with which the DNA occursin natural sources. These DNA sequences, including the sequencesreported in FIGS. 1A to 1H [SEQ ID NO: 1 and 2] and identified above,code for the expression for MSF polypeptides. These sequences, whenexpressed in mammalian cells, yield precursor MSFs which are processedin the mammalian cells to yield functional proteins. Similar processingis expected to be seen in other non-mammalian expression systems.

Examples of MSF DNA sequences may include a DNA sequence comprisingnucleotides 1 through 390 of FIG. 1A [SEQ ID NO: 85]. Another MSF DNAsequence comprises nucleotides 1 through 423 of FIG. 1A [SEQ ID NO: 79].Another MSF DNA sequence comprises nucleotides 1 through 516 of FIG. 1A[SEQ ID NO: 87]. Yet another example of an MSF DNA sequence comprisesnucleotides 1 through 576 of FIG. 1A [SEQ ID NO: 89]. Still a furtherillustration of an MSF DNA sequence comprises nucleotides 1 through 612of FIGS. 1A to 1H [SEQ ID NO: 91]. An additional MSF DNA sequencecomprises nucleotides 1 through 627 of FIGS. 1A to 1B [SEQ ID NO: 93].An MSF DNA sequence may also comprise nucleotides 1 through 660 of FIGS.1A to 1B [SEQ ID NO: 95].

Other MSF DNA sequences include the 5′ sequences of certain alternatelyspliced MSFs, such as a sequence [SEQ ID NO: 109] comprising nucleotides1-76 of FIG. 1A [SEQ ID NO: 3] fused in frame to nucleotides 200-319 ofFIG. 1A [SEQ ID NO: 7], fused in frame to nucleotides 598-748 of FIG. 1B[SEQ ID NO: 111]. Another such 5′ DNA sequence [SEQ ID NO: 113 and 114]comprises nucleotides 1-319 of FIG. 1A [SEQ ID NO: 55] fused in frame tonucleotides 598-748 of FIG. 1B [SEQ ID NO: 111]. Still another DNAsequence [SEQ ID NO: 115] comprising nucleotides 1-469 of FIG. 1A [SEQID NO: 117] fused in frame to nucleotides 598-748 of FIG. 1B [SEQ ID NO:111]. Another MSF DNA sequence [SEQ ID NO: 119] comprises nucleotides1-76 of FIG. 1A [SEQ ID NO: 3] fused in frame to nucleotides 200-319 ofFIG. 1A [SEQ ID NO: 7], fused in frame to nucleotides 598-748 of FIG. 1B[SEQ ID NO: 111]. Still another DNA sequence [SEQ ID NO: 117] extendsfrom nucleotides 1 through 469 of FIG. 1A. Another MSF DNA sequence [SEQID NO: 121] comprises nucleotides 1 to 76 of FIG. 1A [SEQ ID NO: 3]fused in frame to nucleotides 200 through 469 of FIG. 1A [SEQ ID NO:7-9].

Other MSF DNA sequences which encode homo- or heterodimers of theabove-described MSF DNA sequences or DNA sequences encoding abiologically active fragment of such sequences are also included in thisinvention. Similarly an allelic variation of the MSF DNA sequences, anda DNA sequence capable of hybridizing to any of MSF DNA sequences, whichencodes a peptide or polypeptide having activity in the fibrin clotassay are also encompassed by this invention.

It is understood that the DNA sequences of this invention which encodebiologically active human MSFs may also comprise DNA sequences capableof hybridizing under appropriate conditions, or which would be capableof hybridizing under said conditions, but for the degeneracy of thegenetic code, to an isolated DNA sequence of FIGS. 1A to 1H [SEQ ID NO:1 and 2] or to active MSFs formed by alternate splicing of two or moreexons of FIGS. 1A to 1H as described above. These DNA sequences includethose sequences encoding all or a fragment of the above-identified exonpeptide sequences and those sequences which hybridize under stringent orrelaxed hybridization conditions [see, T. Maniatis et al, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982),pages 387 to 389] to the MSF DNA sequences. An example of one suchstringent hybridization condition is hybridization in 4×SSC at 65° C.,followed by a washing in 0.1×SSC at 65° C. for an hour. Alternatively anexemplary stringent hybridization condition is in 50% formamide, 4×SSCat 50° C.

DNA sequences which hybridize to the sequences for an MSF under relaxedhybridization conditions and which code for the expression of MSFpeptides having MSF biological properties also encode novel MSFpolypeptides. Examples of such non-stringent hybridization conditionsare 4×SSC at 50° C. or hybridization with 30-40% formamide at 42° C. Forexample, a DNA sequence which shares regions of significant homology,e.g., Exons II [SEQ ID NO: 5], III [SEQ ID NO: 7] or IV [SEQ ID NO: 9],and/or glycosylation sites or disulfide linkages, with the sequences ofMSF and encodes a protein having one or more MSF biological propertyclearly encodes an MSF polypeptide even if such a DNA sequence would notstringently hybridize to the MSF sequences.

The DNA sequences of this invention may include or contain modificationsin the non-coding sequences, signal sequences or coding sequences basedon allelic variation among species, degeneracies of the genetic code ordeliberate modification. Allelic variations are naturally-occurring basechanges in the species population which may or may not result in anamino acid change. Degeneracies in the genetic code can result in DNAsequences which code for MSF polypeptides but which differ in codonsequence. Deliberate modifications can include variations in the DNAsequence of MSF which are caused by point mutations or by inducedmodifications to enhance the activity, half-life or production of thepolypeptides encoded thereby. All such sequences are encompassed in theinvention.

Utilizing the sequence data in FIGS. 1A to 1H [SEQ ID NO: 1 and 2] andthe exon combinations described above, as well as the denotedcharacteristics of MSF, it is within the skill of the art to modify DNAsequences encoding an MSF and the resulting amino acid sequences of MSFby resort to known techniques.

Modifications of interest in the MSF sequences may include thereplacement, insertion or deletion of a selected nucleotide or aminoacid residue in the coding sequences. For example, the structural genemay be manipulated by varying individual nucleotides, while retainingthe correct amino acid(s), or the nucleotides may be varied, so as tochange the amino acids, without loss of biological activity. Mutagenictechniques for such replacement, insertion or deletion, e.g., in vitromutagenesis and primer repair, are well known to one skilled in the art[See, e.g., U.S. Pat. No. 4,518,584]. The encoding DNA of a naturallyoccurring MSF may be truncated at its 3′-terminus while retaining itsbiological activity. A recombinant, genetically-engineered MSF DNAsequence may be altered or truncated at both its 3′ and 5′ termini whileretaining biological activity. It also may be desirable to remove theregion encoding the signal sequence, and/or to replace it with aheterologous sequence. It may also be desirable to ligate a portion ofthe MSF sequence to a heterologous coding sequence, and thus to create afusion peptide with the biological activity of MSF.

Specific mutations of the sequences of an MSF polypeptide may involvemodifications of a glycosylation site. The absence of glycosylation oronly partial glycosylation results from amino acid substitution ordeletion at any asparagine-linked glycosylation recognition site or atany site of the molecule that is modified by addition of O-linkedcarbohydrate. An asparagine-linked glycosylation recognition sitecomprises a tripeptide sequence which is specifically recognized byappropriate cellular glycosylation enzymes. These tripeptide sequencesare either Asn-X-Thr or Asn-X-Ser, where X can be any amino acid exceptproline. For example, such a site can be found in the cDNA illustratedin FIGS. 1A to 1H [SEQ ID NO: 2] at amino acids #206-#208. A variety ofamino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Expression of such alterednucleotide sequences produces variants which are not glycosylated atthat site.

Other analogs and derivatives of the sequence of an MSF which would beexpected to retain MSF activity in whole or in part may also be easilymade by one of skill in the art given the disclosures herein. One suchmodification may be the attachment of polyethylene glycol (PEG) ontoexisting lysine residues in an MSF sequence, as taught in U.S. Pat. No.4,904,584, which is incorporated herein by reference. Alternatively, theinsertion of one or more lysine residues or other amino acid residuesthat can react with PEG or PEG derivatives into the sequence byconventional techniques may enable the attachment of PEG moieties.Existing cysteines may be used according to techniques taught inpublished PCT Patent Application US90/02144, which is incorporatedherein by reference. Such modifications are believed to be encompassedby this invention.

In addition to the above, other open reading frames (ORFs) or structuralgenes encoding MSFs may be obtained and/or created from cDNA librariesfrom other animal cell sources. For example, a murine MSF genomic cloneand several partial MSF cDNA clones have been isolated by the inventors.

A naturally occurring MSF of this invention may be obtained as a singlehomogeneous protein or mixture of various alternately spliced MSFproteins and purified from natural sources. Among such natural sourcesare human urine or stimulated PBLs, other mammalian cell sourcesproducing the factors naturally or upon induction with other factorsfrom cell lines. The DNA of such MSFs may also be obtained and purifiedfrom natural sources.

To isolate and purify the naturally-occurring MSFs from natural sources,the purification technique comprises the following steps which aredescribed in more detail in Example 1 below. The example. and thefollowing summary illustrate the purification for an exemplarynaturally-occurring MSF, human urinary meg-CSF, which is isolated fromhuman urine. For the urinary meg-CSF, the purification includesconcentrating pooled bone marrow transplant patient urine through anAmicon YM-10 filter. The concentrated urine is passed through an anionexchange chromatographic column and the flow-through is bound onto acation exchange chromatographic column. The urinary protein eluate isthen subjected to pooling, dialyzing and heating and is applied to alectin affinity chromatographic column. This eluate is then dialyzed andapplied to a cation exchange fine performance liquid chromatography(FPLC) column. Finally this eluate is applied through three cycles ofreverse phase high pressure liquid chromatography (HPLC) using differentsolvent systems for each HPLC run.

According to this purification scheme, batches with the highest levelsof MSF in the murine fibrin clot assay, described below, are selectedfor further purification at the semi-preparative scale (between 30 and100 liters urine equivalent) to maximize recovery and yield. Thus ahomogeneous MSF, native meg-CSF, may be obtained by applying thepurification procedures described in Example 1 to human urine or othersources of human MSF, e.g., activated PBLs.

Other tissue sources and cell lines from which natuially occurring MSFsmay be isolated include HeLa cell lines, e.g. ATCC #098-AH2, and bonemarrow cell lines, such as murine bone marrow cell line, FCM-1 [GeneticsInstitute, Inc., Cambridge, Mass.], osteosarcoma cell line U20S, smallcell lung carcinoma H128, neuroblastoma SK-N-SH, neuroblastoma SK-N-MC,neuroglioma epithelial-like cell line H4, erythroleukemia cell lineOCIM1 and OCIM2, erythroleukemia cell line K562 in the presence of PMA,erythroleukemia cell line HEL in the presence of PMA, and hepatoma cellline HEPG2. Procedures for culturing a cell source which may be found toproduce an MSF are known to those of skill in the art.

The MSF proteins and the DNA sequences encoding MSFs of this inventioncan be produced via recombinant genetic engineering techniques andpurified from a mammalian cell line which has been designed to secreteor express the MSF to enable large quantity production of pure, activeMSFs useful for therapeutic applications. The proteins may also beexpressed in bacterial cells, e.g., E. coli, and purified therefrom. Theproteins may also be expressed and purified in yeast cells or inbaculovirus or insect cells. Alternatively, an MSF or active fragmentsthereof may be chemically synthesized. An MSF may also be synthesized bya combination of the above-listed techniques. Suitable techniques forthese different expression systems are known to those of skill in theart.

To produce a recombinant MSF, the DNA sequence encoding the factor canbe introduced into any one of a variety of expression vectors to make anexpression system capable of producing an MSF or one or more fragmentsthereof in a selected host cell.

The DNA sequences of the individual exons may be obtained by chemicalsynthesis or may be obtained from the following two deposits by standardrestriction enzyme subcloning techniques or by the polymerase chainreaction (PCR) using synthetic primers for each exon based on thenucleotide sequences of FIGS. 1A to 1H [SEQ ID NO: 1]. Two genomicclones containing meg-CSF sequences which may be used as sources of theMSF sequences have been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, USA inaccordance with the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure on Aug. 3, 1990.

An approximately 12 kb genomic fragment (referred to as Meg Kpn-SnaBI)containing the sequences spanning Exon I through part of Exon VI [SeeFIG. 2, the 5′ KpnI site to the 3′ SnaBI site] in an E. coli plasmid wasgiven the accession number ATCC 40857. As described hereinbefore, theentire 18.2 kb sequence of spanning Exons I through Exon X (referred toas 18-5665) [SEQ ID NO: 3-22] inserted into bacteriophage lambda DNA wasdeposited under the accession number ATCC 40856. Exons XI and XII [SEQID NO: 13 and 25] may be made from the PBL derived cDNA clone stored atGenetics Institute, Inc., referred to above.

The MSF DNA obtained as described above or modified as described abovemay be introduced into a selected expression vector to make arecombinant molecule or vector for use in the method of expressing novelMSF polypeptides. These vectors contain the novel MSF DNA sequencesrecited herein, which alone or in combination with other sequences, codefor MSF polypeptides of the invention or active fragments thereof. Thevector employed in the method also contains selected regulatorysequences in operative association with the DNA coding sequences of theinvention. Regulatory sequences preferably present in the selectedvector include promoter fragments, terminator fragments and othersuitable sequences which direct the expression of the protein in anappropriate host cell. The resulting vector is capable of directing thereplication and expression of an MSF in selected host cells. Thetransformation of these vectors into appropriate host cells can resultin expression of the MSF polypeptides.

Numerous types of appropriate expression vectors are known in the artfor mammalian (including human) expression, as well as insect, yeast,fungal and bacterial expression, by standard molecular biologytechniques. Mammalian cell expression vectors are desirable forexpression. Bacterial cells, e.g., E. coli, are also desirable forexpression of MSFs.

Mammalian cell expression vectors described herein may be synthesized bytechniques well known to those skilled in this art. The components ofthe vectors, e.g. replicons, selection genes, enhancers, promoters, andthe like, may be obtained from natural sources or synthesized by knownprocedures. See, Kaufman et al, J. Mol. Biol., 159:511-521 (1982); andKaufman, Proc. Natl. Acad. Sci., USA, 82:689-693 (1985). Alternatively,the vector DNA may include all or part of the bovine papilloma virusgenome [Lusky et al, Cell, 36:391401 (1984)] and be carried in celllines such as C127 mouse cells as a stable episomal element.

One such vector for mammalian cells is pXM [Y. C. Yang et al, Cell,47:3-10 (1986)]. This vector contains the SV40 origin of replication andenhancer, the adenovirus major late promoter, a cDNA copy of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,in appropriate relationships to direct the high level expression of thedesired cDNA in mammalian cells [See, e.g., Kaufman, Proc. Natl. Acad.Sci. USA, 82:689-693 (1985)]. To generate constructs for expression ofMSF, the pXM vector is linearized with an appropriate restrictionendonuclease enzyme and separately ligated to the cDNA encoding MSFwhich has been appropriately prepared by restriction endonucleasedigestion, for example.

Another similar vector is pMT21. pMT21 is prepared by linearizing pMT2pc(which has been deposited with the ATCC under Accession Number 40348) bydigestion with PstI. The DNA is then blunted using T₄ DNA polymerase. Anoligonucleotide [SEQ ID NO: 123]:

TGCAGGCGAG CCTGAATTCC TCGA 24is then ligated into the DNA, recreating the PstI site at the 5′ end andadding an EcoRI site and XhoI site before the ATG of the DHFR cDNA. Thisplasmid is called pMT21. Preferably a desired polylinker withrestriction sites for NotL KpnI, SalI and SnabI is introduced into thisvector for ready insertion of the MSF coding sequence.

Still another vector which may be employed to express MSF in CHO cellsis pED4DPC-1. This vector is prepared from pED4, also known as pEMC2B 1.As does pXM, described above, this vector contains the SV40 origin ofreplication and enhancer, the adenovirus major late promoter, a cDNAcopy of the majority of the adenovirus tripartite leader sequence, asmall hybrid intervening sequence, an SV40 polyadenylation signal andthe adenovirus VA I gene, in appropriate relationships to direct thehigh level expression of the desired cDNA in mammalian cells. Inaddition, it contains DHFR and β-lactamase markers and an EMC sequencewhich pXM does not contain. To made pED4DPC-1, the sequence 1075 through1096 is removed from pED4 to remove a stretch of cytosines. A newpolylinker is added to introduce the restriction sites NotI, SalI andSnabI to the plasmid. The vector is linearized with an appropriateendonuclease enzyme and subsequently ligated separately to the cDNAencoding MSF.

These above-described vectors do not limit this invention; because oneskilled in the art can also construct other useful mammalian expressionvectors by, e.g., inserting the DNA sequence of the MSF from the plasmidwith appropriate enzymes and employing well-known recombinant geneticengineering techniques and other known vectors, such as pJL3 and pJL4[Gough et al., EMBO J., 4:645-653 (1985)] and pMT2 (starting withpMT2-VWF, ATCC #67122; see PCT application PCT/US87/00033).

Once the vector is prepared, a selected host cell is transformed byconventional techniques with the vector containing MSF. The method ofthis present invention therefore comprises culturing a suitable cell orcell line, which has been transformed with a DNA sequence coding forexpression of an MSF polypeptide under the control of known regulatorysequences.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or the monkey COS-1 cell line. CHO cells arepreferred as a mammalian host cell of choice for stable integration ofthe vector DNA, and for subsequent amplification of the integratedvector DNA, both by conventional methods. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625(1981), or alternatively, Kaufman et al, Mol. Cell. Biol.,5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Anothersuitable mammalian cell line is the CV-1 cell line. Further exemplarymammalian host cells include particularly primate cell lines and rodentcell lines, including transformed cell lines. Normal diploid cells, cellstrains derived from in vitro culture of primary tissue, as well asprimary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude, but are not limited to, HeLa, mouse L-929 cells, 3T3 or 293lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, MC1061 and strains used in the following examples) are well-knownas host cells in the field of biotechnology. When used as host cells, E.coli permits the expression of the MSF protein as a single protein. MSFmay also be expressed in bacterial cells as a fusion protein withthioredoxin, as disclosed in detail in U.S. patent application Ser. No.07/652,53 1, which is incorporated herein by reference. Various strainsof B. subtilis, Pseudomonas, other bacilli and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein. Fungal cells may also be employed asexpression systems.

Once the MSF is expressed by the transformed and cultured cells, it isthen recovered, isolated and purified from the culture medium (or fromthe cell, if expressed intracellularly) by appropriate means known toone of skill in the art.

A preferred purification procedure to isolate a recombinant or syntheticMSF from serum free mammalian cell (COS-1) conditioned medium ischaracterized by steps are similar to those for the purification ofnative meg-CSF from urine and are described in detail in Example 7. Therecombinant MSF is concentrated from COS-1 cell supernatant through anAmicon YM-10 filter with a 10,000 Dalton molecular weight cut-off. Theconcentrate is dialyzed into 20 mm sodium acetate, pH4.5, and thenapplied to an S Toyopearl cation exchange FPLC column equilibrated in 20mM sodium acetate, pH4.5. The bound material is then eluted from thecolumn and applied through a cycle of C4 reverse phase HPLC using 0.1%TFA/acetonitrile as the solvent system. In the case of MSF-K130, theprotein elutes between 20-30% of a buffer containing 0.1% TFA, 95%acetonitrile. Other non-naturally occurring MSFs described above may beobtained by applying this purification scheme described in detail inExample 7 for MSF-K130 [SEQ ID NO: 77 and 78].

MSF polypeptides may also be produced by known conventional chemicalsynthesis, e.g., by Merrifield synthesis or modifications thereof.Methods for constructing the polypeptides of the present invention bysynthetic means are known to those of skill in the art. Thesynthetically-constructed MSF polypeptide sequences, by virtue ofsharing primary, secondary, or tertiary structural and conformationalcharacteristics with native MSF polypeptides may possess MSF biologicalproperties in common therewith. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedMSF polypeptides in therapeutic and immunological processes.

One or more MSFs or active fragments thereof, purified in a homogeneousform or as a mixture of different MSFs from natural sources or producedrecombinantly or synthetically, may be used in a pharmaceuticalpreparation or formulation. The MSF pharmaceutical compositions of thepresent invention or pharmaceutically effective fragments thereof may beemployed in the treatment of immune deficiencies or disorders. MSFs mayalso be employed to treat deficiencies in hematopoietic progenitor orstem cells, or disorders relating thereto. MSFs may be employed inmethods for treating cancer and other pathological states resulting fromdisease, exposure to radiation or drugs, and including for example,leukopenia, bacterial and viral infections, anemia, B cell or T celldeficiencies, including immune cell or hematopoietic cell deficiencyfollowing a bone marrow transplantation. MSFs may also be used topotentiate the immune response to a variety of vaccines creating longerlasting and more effective immunity. MSFs may be employed to stimulatedevelopment of B cells, as well as megakaryocytes.

The MSFs of the present invention may also have utility in stimulatingplatelet production, stimulating platelet recovery followingchemotherapy or bone marrow transplantation, treating thrombocytopenia,aplastic anemia and other platelet disorders, preserving and extendingthe lifetime of platelets in storage, and stimulating plateletproduction in vitro for use in platelet transfusions. MSFs may also beemployed to stimulate the growth and development of other colonies ofhematopoietic and non-hematopoietic cells. Similarly, these factors maybe useful in cell-targeting. MSF may also be useful in the treatment ofwounds or bums, alone or with other wound-healing agents, such asfibroblast growth factor (FGF). Adhesion related therapeutic uses arealso contemplated for MSFs of this invention. MSF compositions may beused as an adjunctive therapy for bone marrow transplant patients.

Therapeutic treatment of such platelet disorders cr deficiencies withthese MSF polypeptide compositions may avoid undesirable side effectscaused by treatment with presently available serum-derived factors ortransfusions of human platelets. It may also be possible to employ oneor more active peptide fragments of MSF in such pharmaceuticalformulations.

Therefore, as yet another aspect of the invention are therapeuticcompositions for treating the conditions referred to above. Suchcompositions comprise a therapeutically effective amount of a MSFprotein, a therapeutically effective fragment thereof, or a mixture ofvariously spliced or otherwise modified MSFs in admixture with apharmaceutically acceptable carrier. This composition can besystemically administered parenterally. Alternatively, the compositionmay be administered intravenously. If desired, the composition may beadministered subcutaneously. When systemically administered, thetherapeutic composition for use in this invention is in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, having due regardto pH, isotonicity, stability and the like, is within the skill of theart.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician consideringvarious factors which modify the action of drugs, e.g. the condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. Generally, the dailyregimen should be in the range of about 1 to about 1000 micrograms ofMSF protein, mixture of MSF proteins or fragments thereof. Alternativelyabout 50 to about 50,000 units (i.e., one unit being the minimumconcentration of MSF protein, or MSF protein mixture, which yields themaximal number of colonies in the murine fibrin clot megakaryocytecolony formation assay) of MSF protein per kilogram of body weight maybe a desirable dosage range.

The therapeutic method, compositions, purified proteins and polypeptidesof the present invention may also be employed, alone or in combinationwith other cytokines, hematopoietins, interleukins, growth factors orantibodies in the treatment of disease states characterized by othersymptoms as well as platelet deficiencies. It is anticipated that anMSF, if it does not itself have TPO activity, will prove useful intreating some forms of thrombocytopenia in combination with generalstimulators of hematopoiesis, such as IL-3, IL-6, GM-CSF, steel factor,IL-11 (described in co-owned copending U.S. patent application Ser. No.07/526,474 and in published PCT Patent Application No. WO91/07495, bothincorporated herein by reference) or with other megakaryocyticstimulatory factors or molecules with TPO-like activity. Additionalexemplary cytokines or hematopoietins for such co-administration includeTPO, G-CSF, the CSF-1s (including M-CSF), IL-1, IL-4, IL-7,erythropoietin, and variants of all of these cytokines, or a combinationof multiple cytokines. The dosage recited above would be adjusted tocompensate for such additional components in the therapeuticcomposition. For example, the MSF may be administered in amounts from 1to 1000 μg/kg body weight and the other cytokine may be administered inthe same amounts in such a co-administration protocol. Alternatively,co-administration may permit lesser amounts of each therapeutic agent tobe administered. Progress of the treated patient can be monitored byconventional methods.

Other uses for these novel proteins and recombinant polypeptides are inthe development of antibodies generated by standard methods for in vivoor in vitro diagnostic or therapeutic use. As diagnostic or researchreagents, antibodies generated against these MSFs may also be useful inaffinity columns and the like to further purify and identify thecomplete meg-CSF protein. Such antibodies may include both monoclonaland polyclonal antibodies, as well as chimeric antibodies or“recombinant” antibodies generated by known techniques.

The antibodies of the present invention may be utilized for in vivo andin vitro diagnostic purposes, such as by associating the antibodies withdetectable labels or label systems. Alternatively these antibodies maybe employed for in vivo and in vitro therapeutic purposes, such as byassociation with certain toxic or therapeutic compounds or moities knownto those of skill in this art. These antibodies also have utility asresearch agents.

Also provided by this invention are the cell lines generated bypresenting MSF or a fragment thereof as an antigen to a selected mammal,followed by fusing cells of the animal with certain cancer cells tocreate immortalized cell lines by known techniques. The methods employedto generate such cell lines and antibodies directed against all orportions of a human MSF protein or recombinant polypeptide of thepresent invention are also encompassed by this invention.

The following examples illustratively describe the purification andcharacteristics of homogeneous human MSF and other methods and productsof the present invention. These examples are for illustration and arenot intended to limit the scope of the present invention.

EXAMPLE 1 Purification of Native meg-CSF from Urine

The following procedures are employed to obtain native meg-CSF proteinfrom urine of human bone marrow transplant patients. The same or similarprocedure may be employed to purify other MSFs from natural sources.Urine from patients with aplastic anemia or thrombocytopeniaaccompanying other disease states may also be used as the source of thefactor employing this purification.

STEP 1: Urine was collected from bone marrow transplant patients betweendays 5 and 18 following transplant. One hundred liters of pooled urinewere treated with protease inhibitors phenylmethyl-sulfonylfluoride(PMSF) and ehtylenediaminetetraacetic acid (EDTA). This pooled urine wasconcentrated on an Amicon YM-10 filter (10,000 molecular weight cut-off)to remove excess pigments and reduce the volume. A cocktail of proteaseinhibitors [leupeptin, aprotinin, ethylene glycol-bis-tetraacetic acid(EGTA) and N-ethylmaleimide (NEM)] was added to the urine at this stepand the next three steps to minimize proteolysis. The pH of the urineconcentrate was adjusted to 8.0 and diluted to a conductivity of 7mS/cm.

STEP 2: The retentate from this first step of the purification was thensubjected to anion exchange column chromatography on a QAE Zetaprep[Cuno] at pH 8.0. The QAE flow-through was adjusted to a pH4.5 with 1Macetic acid.

STEP 3: The flow-through from the second purification step was bound toa cation exchange chromatographic column, an SP-Zetaprep column [Cuno]at pH 4.5. Bound protein containing Meg-CSF was eluted with 1M NaCl at apH of 4.5. The eluate was pooled, protease inhibitors were added asabove and the bound protein was either neutralized to pH7 and stored at−80° C. until further chromatography was performed or dialyzed into aTris-buffered saline (TBS) solution, with the addition of the proteaseinhibitors described in Step 1. This dialyzate was heated at 56° C. for30 minutes. Addition of the protease inhibitors, while not essential forrecovery of protein, enabled greater amount of protein to be recoveredfrom this step, not denatured by the proteases in the system. Pools fromthis step were also analyzed for the presence of megakaryocyte-specificgrowth factors. These pools were found to contain Meg-CSF activity.

STEP 4: The resulting material was added to a lectin affinitychromatographic column, a Wheat Germ Sepharose column [Pharmacia].Urinary Meg-CSF was found to bind to this column. This protein was theneluted with 0.25 M N-acetyl glucosamine (N-acglcNH₂) in TBS, anddialyzed against 20 mM sodium acetate, pH 4.5 in the presence of theprotease inhibitors of Step 1.

STEP 5: This dialysate was applied to a 10 ml S-Toyopearl FPLC cationexchange column and eluted using a linear gradient of 0 to 1M NaCl in 20mM sodium acetate at pH 4.5. The protein eluted from this step wastested for Meg-CSF activity in the fibrin clot assay described below.The Meg-CSF activity was observed to elute in two discrete peaks. Themajor activity eluted between 0.1M and 0.25M NaCl. A minor, butreproducible activity eluted between 0.3M and 0.5N NaCl. The twoactivities may be due to protein or carbohydrate modification of asingle protein; however the data presented further herein refers to themajor protein.

STEP 6: The eluate from this fifth purification step was then purifiedon a reverse phase HPLC (C4) column [Vydac; 1 cm×25 cm] which was elutedwith a linear gradient of between 23-33% acetonitrile in 0.1%trifluoroacetic acid (TFA). This step removes an abundant 30Kd proteincontaminant. Recombinant MSF elutes at a slightly lower gradient ofabout 20 to about 30% of the buffer.

STEP 7: The HPLC step was repeated in a different solvent system, afterthe eluate of Step 6 was diluted with two parts acetic acid andpyridine. The purified material eluted between 6-15% n-propanol inpyridine and acetic acid on a C18 reverse phase HPLC column (0.46×25cm). The material produced after this step, when assayed gave thespecific activity of greater than 5×107 dilution units reported in themurine assay. This optional step removes the bulk of urinaryribonuclease, a major contaminant, from the preparation.

STEP 8: The HPLC step was repeated once more on a C4 column (Vydac;0.46×25 cm) using 0.15% HFBA in acetonitrile. The material elutedbetween 27-37% acetonitrile. The last HPLC step removed substantiallyall remaining ribonuclease and proteinaceous contaminants present afterStep 7.

This purified meg-CSF material was then analyzed by. SDS-PAGE,bioassayed and labelled with ¹²⁵I. Homogenous protein is obtained fromthis procedure, omitting step 7, having a specific activity ranging fromabout 5×10⁷ to about 2-5×10⁸ dilution units per mg protein in the murinemegakaryocyte colony assay described below. A unit of activity isdefined as the reciprocal of the maximum dilution which stimulates themaximum number of megakaryocyte colonies per ml.

This process is preferably used to purify any naturally occurring MSFprotein, or mixture thereof, from a natural source.

EXAMPLE 2 Analysis of Genomic MSF, meg-CSF

A preliminary analysis of COS supernatant expressing the Kpn-SnaB1 12 kbgenomic subclone was performed previously, and indicated that a proteinwhich reacted with MSF-specific antibodies was expressed by COS cellsand was secreted into the culture medium. Dialysed, concentrated cellsupernatant was variably active in the murine meg-colony assay.

Analysis by Northern and Western indicated that the level of MSF mRNAand protein was very low. A Western immunoblot of the protein from COSsupernatant expressed in conditioned medium revealed the presence ofthree heterogenous species which specifically bind anti-meg CSF peptideantibodies and the binding can be competed with excess peptide. Themolecular weights of these species, 200 kD, 30 kD, and 14 kD, aredifferent from the partially purified meg-CSF from the BMT urine whichhas an apparent molecular weight ranging between about 18-about 28 kDunder reducing conditions.

EXAMPLE 3 Mammalian Cell Expression of MSF-K130

Recombinant MSFs were obtained by the following techniques. Six MSF cDNAclones, truncated at putative MSF protein processing sites in Exons Vand VI, were constructed by using the polymerase chain reaction. Aseventh clone, MSF-K130 [SEQ ID NO: 77 and 78], was isolated as aconsequence during the PCR reaction by the inadvertent insertion of anartificial termination codon in Exon IV after amino acid 130. Sevenoligonucleotide primers were synthesized as follows:

(1) CGCGCGGCCGCGACTATTCG [SEQ ID NO: 124] (2) GCGCTCGAGCTAAGAGGAGGAGGA[SEQ ID NO: 125] (3) GCGCTCGAGCTATCTATTAGCAGC [SEQ ID NO: 126] (4)GCGCTCGAGCTACTTGTTATCTTT [SEQ ID NO: 127] (5) GCGCTCGAGCTAATCTACAACTGG[SEQ ID NO: 128] (6) GCGCTCGAGCTAGTTTGGTGGTTT [SEQ ID NO: 129] (7)GCGCTCGAGCTAAGTTCTGTTCTT [SEQ ID NO: 130]

Primer (1) was designed to hybridize to the cDNA flanking the initiatingmethionine codon and contains nine MSF-homologous nucleotides, a NotIrestriction endonuclease site and three additional nucleotides toenhance restriction endonuclease recognition (as suggested in the NewEngland Biolabs catalog).

The remaining oligonucleotide primers were designed to hybridize to the3′ regions of the cDNA and to flank the putative MSF protein processingsite codons for [SEQ ID NO: 87] MSF-S172 (2), [SEQ ID NO: 89] MSF-R192(3), [SEQ ID NO: 91] MSF-K204 (4), [SEQ ID NO: 77] MSF-K130 and [SEQ IDNO: 95] D220 (5), [SEQ ID NO: 79] N141 (6) and [SEQ ID NO: 131] T208(7). The 3′ primers contain twelve nucleotides of MSF-homologoussequence, a translation termination codon, an XhoI restrictionendonuclease site and three additional nucleotides to enhancerestriction endonuclease recognition.

Six PCR reactions were performed in duplicate, using the conditionsrecommended by Perkin-Elmer Cetus Corp. Each of the six duplicatereactions contained the 5′ primer (No. 1, 1.0 μM), one of the 3′ primers(1.0 μM) and 1 ng of MSF cDNA as template. The reactions were carriedthrough two rounds of eighteen cycles each. One cycle consisted of a twominute denaturation of 95° C. followed by three minutes ofannealing/extension of 72° C. After the first round of eighteen cycles,10 μl of the first reaction was transferred to a fresh reaction mixture(100 μl total), and the amplification cycles were repeated. The PCRproducts generated by the second round of amplification reactions(twelve in all) were digested with NotI and XhoI, using conditionsdescribed by the vendor, and fractionated by agarose gelelectrophoresis.

To obtain expression of these truncated MSFs in mammalian host cells,the appropriate DNA bands were then excised and ligated into a NotI andXhoI digested pMT21-2 vector. The vector pMT21-2 is identical to thevector pMT21 except for the polylinker region, containing PstI, NotI,KpnI, Apal, EcoRV, EcoRI and XhoI sites, which was changed to facilitatecloning of MSF DNA fragments. Competent DH5 cells were transformed withthe recombinant plasmid and selected for resistance to ampicillin.Plasmid DNA was prepared from transformed cells and sequenced withselected internal oligonucleotides across the MSF insert. All the abovetechniques are conventional and described in Sambrook et al, citedabove.

The clones listed above were identified as having the correct nucleotidesequence to encode the desired MSF proteins. For example, S172 [SEQ IDNO: 87] encodes MSF amino acids 1-172 [SEQ ID NO: 88], terminating witha serine residue. Position 173 encodes a translation termination codon.The exception was one of the two reactions designed to synthesize D220[SEQ ID NO: 95 and 96]. During this PCR reaction, a serendipitousdeletion of nucleotide 392 of the cDNA sequence resulted in cloneMSF-K130 [SEQ ID NO: 77], which encodes MSF amino acids 1-130 [SEQ IDNO: 78] and terminates in a lysine followed by a TAA stop codon. CloneMSF-K130 [SEQ ID NO: 77 and 78] may readily be deliberately synthesizedby a PCR reaction designed for this purpose. This would require using anoligonucleotide primer similar in design to the other 3′ primeroligonucleotides, i.e., an oligonucleotide containing twelve nucleotidesof MSF-homologous sequence, a translation termination codon, an XhoIrestriction site and a few additional nucleotides to enhance restrictionendonuclease recognition. An example of a suitable 3′ primer would bethe following sequence: SEQ ID NO: 133 GCGCTCGAGCTAATTTGATGGTTT.

The pMT21-2 plasmid, containing the MSF DNA sequence is transfected ontoCOS cells. The conditioned medium from the transfected COS cellscontains MSF biological activity as measured in the murine assays.Similarly the modified pED4DPC-1 construct containing the cDNA for MSFis transfected into CHO cells.

The vector pED4DPC-1 may be derived from pMT21 vector. pMT21 is cut withEcoRI and XhoI which cleaves the plasmid at two adjacent cloning sites.An EMCV fragment of 508 base pairs is cut from pMT₂ECAT₁ [S. K. Jong etal, J. Virol., 63:1651-1660 (1989)] with the restriction enzymes EcoRIand TaqαI. A pair of oligonucleotides 68 nucleotides in length aresynthesized to duplicate the EMCV sequence up to the ATG. The ATG ischanged to an ATT, and a C is added, creating a XhoI site at the 3′ end.A TagαI site is situated at the 5′ end. The sequences of theoligonucleotides are [SEQ ID NO: 134]:

CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC 40 GTGGTTTTCC TTTGAAAAACACGATTGC 68and the respective complementary strands.

Ligation of the pMT21 EcoRI-to-XhoI fragment to the EMCV EcoRI-to-TaqαIfragment and to the TaqαI/XhoI oligonucleotides produces the vectorpED4. A polylinker, containing PstI, NotI, SalI, SnaBI and EcoRI sites,is inserted into this vector as described above to create pED4PC-1.

Stable transformants are then screened for expression of the product bystandard immunological, biological or enzymatic assays, such as thosedescribed below in Example 8. The presence of the DNA and mRNA encodingthe MSF polypeptides is detected by standard procedures such as Southernand Northern blotting. Transient expression of the DNA encoding thepolypeptides during the several days after introduction of theexpression vector DNA into suitable host cells is measured withoutselection by activity or immunologic assay, e.g., the murine fibrin clotassay, of the proteins in the culture medium.

EXAMPLE 4 Bacterial Expression Systems

One skilled in the art could manipulate the sequences encoding the MSFpolypeptide by eliminating any human regulatory sequences flanking thecoding sequences, eliminating the mammalian secretory sequence of ExonI, and inserting bacterial regulatory sequences to create bacterialvectors for intracellular or extracellular expression of the MSFpolypeptide of the invention by bacterial cells. The DNA encoding thepolypeptides may be further modified to contain different codons tooptimize bacterial expression as is known in the art.

The sequences encoding the mature MSF are operatively linked in-frame tonucleotide sequences encoding a secretory leader polypeptide permittingbacterial expression, secretion and processing of the mature MSFpolypeptides, also by methods known in the art. The expression of MSF inE. coli using such secretion systems is expected to result in thesecretion of the active polypeptide. This approach has yielded activechimeric antibody fragments [See, e.g., Bitter et al, Science,240:1041-1043 (1983)].

Alternatively, the MSF may be expressed as a cytoplasmic protein in E.coli, either directly or as a carboxy terminal fusion to proteins, suchas thioredoxin, which can maintain many peptides in soluble form in E.coli. The fusion proteins can be cleaved and the free MSF isolated usingenzymatic cleavage (enterokinase, Factor Xa) or chemical cleavage(hydroxylamine) depending on the amino acid sequence used to fuse themolecules.

If the cytoplasmic MSF or MSF fusion protein is expressed in inclusionbodies, then either molecule would most likely have to be refolded aftercomplete denaturation with guanidine hydrochloride and a reducing agenta process also known in the art. For procedures for isolation andrefolding of intracellularly expressed proteins, see, for example, U.S.Pat. No. 4,512,922. If either MSF protein or MSF-fusion protein remainin solution after expression in E. coli, they are likely to not requiredenaturation but only mild oxidation to generate the correct disulfidebridges.

The compounds expressed through either route in bacterial host cells maythen be recovered, purified, and/or characterized with respect tophysicochemical, biochemical and/or clinical parameters, all by knownmethods.

EXAMPLE 5 Thioredoxin—MSF Fusion Expression

An MSF can be expressed at high levels in E. coli as a thioredoxinfusion protein using the modified expression vector similar topALtrxA/EK/IL11ΔPro-581 described in pending U.S. patent applicationSer. No. 07/652,351.

As one such example, MSF-K130 [SEQ ID NO: 77 and 78] was employed. Forexpression in E. coli, the first 25 amino acids of Exon I [SEQ ID NO: 3and 4] which encode the secretory leader, were removed from the MSF-K130sequence [SEQ ID NO: 77 and 78]. An enterokinase site, which is [SEQ IDNO: 143] Asp Asp Asp Asp Lys, was inserted at the 5′ end of Exon II [SEQID NO: 5 and 6] of MSF-K130. Additionally, the N-terminal Asp of MSF wasdeleted and replaced with the dipeptide Asn-Gly, encoded by the sequenceAACGGT, which encodes a hydroxylamine cleavage site. The sequence ofMSF-K130 [SEQ ID NO: 77 and 78] which was added as a fusion tothioredoxin, and which contained certain codon changes for preferredexpression in E. coli is shown in FIGS. 3A and 3B [SEQ ID NO: 27 and28].

The plasmid expression vector used for expression is a modifiedpALtrxA/EK/IL11ΔPro-581, illustrated in FIG. 1 of the above-referencedapplication. This plasmid contains the following principal features:

Nucleotides 1-2060 contain DNA sequences originating from the plasmidpUC-18 [Norrander et al, Gene, 26: 101-106 (1983)] including sequencescontaining the gene for β-lactamase which confers resistance to theantibiotic ampicillin in host E. coli strains, and a colE1-derivedorigin of replication. Nucleotides 2061-2221 contain DNA sequences forthe major leftward promoter (pL) of bacteriophage λ [Sanger et al, J.Mol. Biol., 162:729-773 (1982)], including three operator sequences,O_(L)1, O_(L)2 and O_(L)3. The operators are the binding sites for λcIrepressor protein, intracellular levels of which control the amount oftranscription initiation from pL. Nucleotides 2222-2241 contain a strongribosome binding sequence derived from that of gene 10 of bacteriophageT7 [Dunn and Studier J. Mol. Biol., 166:477-535 (1983)].

Nucleotides 2242-2568 contain a DNA sequence encoding the E. colithioredoxin protein [Lim et al, J. Bacteriol., 163:311-316 (1985)].There is no translation termination codon at the end of the thioredoxincoding sequence in this plasmid.

Nucleotides 2569-2583 contain DNA sequence encoding the amino acidsequence for a short, hydrophilic, flexible spacer peptide “—GSGSG—”.Nucleotides 2584-2598 provide DNA sequence encoding the amino acidsequence for the cleavage recognition site of enterokinase (EC 3.4.4.8),“—DDDDK—” [Maroux et al, J. Biol. Chem., 246:5031-5039 (1971)].

Nucleotides 2599-3132 contain DNA sequence encoding the amino acidsequence of a modified form of mature human IL11 [Paul et al, Proc.Natl. Acad. Sci. USA, 87:7512-7516 (1990)], deleted for the N-terminalprolyl-residue normally found in the natural protein. The sequenceincludes a translation termination codon at the 3′-end of the IL11sequence.

Nucleotides 3133-3159 provide a “Linker” DNA sequence containingrestriction endonuclease sites. Nucleotides 3160-3232 provide atranscription termination sequence based on that of the E. coli aspAgene [Takagi et al, Nucl. Acids Res., 13:2063-2074 (1985)]. Nucleotides3233-3632 are DNA sequences derived from pUC-18.

This plasmid is modified in the following manner to replace the ribosomebinding site of bacteriophage T7 with that of λCII. In theabove-described plasmid, nucleotides 2222 and 2241 were removed byconventional means. Inserted in place of those nucleotides was asequence of nucleotides formed by nucleotides 35566 to 35472 and 38137to 38361 from bacteriophage lambda as described in Sanger et al (1982)cited above. This reference is incorporated by reference for the purposeof disclosing this sequence.

The DNA sequence encoding human IL11 in modified pALtrxA/EK/IL11ΔPro-581(nucleotides 2599-3135) is replaced by the sequence shown in FIGS. 3Aand 3B [SEQ ID NO: 27 and 28].

The resulting plasmid was transformed into the E. coli host strain GI724(F⁻, lacI^(q), lacP^(L8), ampC::λcI⁺) by the procedure of Dagert andEhrlich, Gene, 6: 23 (1979). The untransformed host strain E. coli G1724was deposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. on Jan. 31, 1991 under ATCC No. 55151 for patentpurposes pursuant to applicable laws and regulations. Transformants wereselected on 1.5% w/v agar plates containing IMC medium, which iscomposed of M9 medium [Miller, “Experiments in Molecular Genetics”, ColdSpring Harbor Laboratory, New York (1972)] supplemented with 0.5% w/vglucose, 0.2% w/v casamino acids and 100 μg/ml ampicillin.

G1724 contains a copy of the wild-type λcI repressor gene stablyintegrated into the chromosome at the ampC locus, where it has beenplaced under the transcriptional control of Salmonella typhimurium trppromoter/operator sequences. In G1724, λcI protein is made only duringgrowth in tryptophan-free media, such as minimal media or a minimalmedium supplemented with casamino acids such as IMC, described above.Addition of tryptophan to a culture of GI724 will repress the trppromoter and turn off synthesis of λcI, gradually causing the inductionof transcription from pL promoters if they are present in the cell.

GI724 transformed with the MSF containing plasmid was grown at 37° C. toan A₅₅₀ of 0.5 in IMC medium. Tryptophan was added to a finalconcentration of 100 μg/ml and the culture incubated for a further 4hours.

All of the thioredoxin-MSF fusion protein was found in the solublecellular fraction, representing up to 10% of the total protein. Thefusion protein was heat stable, remaining soluble after treatment at 80degrees Celsius for fifteen minutes. Preliminary testing with the fusionprotein has shown activity in the fibrin clot assay described below.

EXAMPLE 6 Other Expression Systems

Manipulations can be performed for the construction of an insect vectorfor expression of MSF polypeptides in insect cells [See, e.g.,procedures described in published European patent application 155,476].

Similarly yeast vectors. may be constructed employing yeast regulatorysequences to express cDNA encoding the precursor, in yeast cells toyield secreted extracellular active MSF. Alternatively the polypeptidemay be expressed intracellularly in yeast, the polypeptide isolated andrefolded to yield active MSF. [See, e.g., procedures described inpublished PCT application WO 86/00639 and European patent application EP123,289.]

EXAMPLE 7 Purification of MSF-K130 from COS Cells

An initial 3 L batch of serum-free conditioned medium from COS-1 cellstransfected with MSF-K130 yielded 140 ug of purified, active MSF proteinusing a three step purification process. COS-1 cell conditioned mediumharvested under serum free conditions was concentrated on an Amicon YM10membrane with a molecular weight cutoff of 10,000 daltons. Theconcentrate was centrifuged at 10,000 rpm in an SS34 rotor at 4° C. toremove cellular debris and precipitate. The supernatant was dialyzedagainst 20 mM sodium acetate pH 4.5 overnight at 4° C. The dialyzedprotein solution was centrifuged again at low speed to remove residualprecipitate.

The dialyzed MSF-K130 containing solution was applied to an S Toyopearlcation exchange FPLC column, equilibrated in 20 mM sodium acetate pH4.5. Bound protein was eluted with a gradient of 0 to 1M NaCl in 20 mMsodium acetate, pH 4.5, at room temperature. Typically, MSF-K130 [SEQ IDNO: 77 and 78] eluted between 0.1 to 0.2 M NaCl. The active MSF peak wasobserved to have a molecular weight on SDS-PAGE 10-20% gradientpolyacrylamide gels of between 20-50 kD under non-reducing conditionsand 18-21 kD under reducing conditions. This was determined by probing aWestern Immunoblot with rabbit antisera raised against a peptide in ExonIII of the MSF gene of FIG. 1A.

The pool of active MSF-K130 [SEQ ID NO: 77 and 78] was divided intothree aliquots based on the molecular weights under non-reducingconditions. Pool A consisted of mostly high molecular weight dimer,35-50 kD. Pool B consisted of intermediate molecular weight dimerspecies ranging from 20-45 kD; and pool C comprised predominantlymonomer species of molecular weight range 14-25 kD. MSF from all threepools had a molecular weight of 18-21 kD under reducing conditions.

The final purification step was one cycle of reverse phase-HPLC. Proteinfrom the three pools were acidified with 10% TFA to 0.1% TFA (v/v),filtered through a 0.45 um PVDF membrane and injected at 1 ml/min onto a25 cm×4.6 mm C4 (Vydec) reverse phase HPLC column equilibrated in 0.1%TFA at room temperature. Bound protein was eluted with a gradient of0-95% acetonitrile in 0.1% TFA. Typically, MSF-K130 activity elutedbetween 15-30% buffer B (95% acetonitrile in 0.1% TFA).

EXAMPLE 8 Biological Activitiesof Human MSFs

The following assays were performed using the purified native urinarymeg-CSF described in Example 1, and in some cases, crude or highlypurified preparations of recombinant MSF-K130 [SEQ ID NO: 77 and 78].The other recombinant or naturally occurring MSFs may exhibit MSFbiological properties in these same assays or other assays.

A. Murine Fibrin Clot Assay

The meg-CSF obtained from Step 7 of the purification techniques ofExample 1 was tested for activity in the megakaryocyte colony formationassay performed substantially as described in S. Kuriya et al, Exp.Hematol., 15:896-901 (1987). A fibrin clot was formed containing 2.5×10⁵mouse bone marrow cells in a 6-well plate. The diluted sample waslayered around the clot and incubated for 6 days. Thereafter, cells werefixed and megakaryocytes were stained for acetylcholinesterase, aspecific marker for murine megakaryocytes. A colony was defined as threeor more megakaryocytes per unit area.

A mixture of pure and mixed colonies containing megakaryocyte colonieswere routinely observed: 70% pure megakaryocyte colonies containing noadditional cell types: 30% mixed megakaryocyte colonies containingadditional non-megakaryocyte cell types to 50% pure: 50% mixed typedepending on the assay. The pure colonies typically contain on average 4to 5 cells per colony, ranging from 3 to 8 cells per colony. The cellswithin the colony are variable in size and appear to contain both matureand immature megakaryocytes. The megakaryocytes are fairly dispersedwithin the colony. A typical mixed megakaryocyte colony is composed onaverage of 10 cells per colony ranging from 7 to 17 cells. The cellsappear more clustered than the megakaryocytes in pure megakaryocytecolonies.

The following control samples were included in every assay. A positivecontrol was WEHI conditioned medium (murine IL-3), which producedbetween 7-25 (average 12) megakaryocyte colonies per clot, approximately50% pure and 50% mixed megakaryocyte colonies. Another positive controlwas serum taken from lethally irradiated dogs at the nadir or low pointof the platelet count [see Mazur et al, Exp Hematol., 13:1164-1172(1985)], which produced between 6-22 (average 15) megakaryocyte coloniesper clot, of which approximately 70% were pure and 30% were mixedmegakaryocyte colonies. The negative control was Iscoves Medium, whichproduced 2-4 megakaryocyte colonies per clot.

In the assay, the purified urinary meg-CSF has a specific activity ofgreater than approximately 5×10⁷ dilution units/mg of protein. A unit ofactivity is defined as described in Example 1.

The major meg-CSF obtained from bone marrow transplant urine eluted fromthe S-Toyopearl cation exchange column chromatography step in thepurification of Example 1 has been analyzed in this assay alone,together, and in combination with other cytokines. In the fibrin clotassay, it produced between 6-16 (average 13) megakaryocyte colonies,with 50-70% pure megakaryocyte colonies. The urinary meg-CSF has beenshown to have variable synergy with murine IL-3 and does not synergizewith human IL-6 or murine IL4 in the fibrin clot culture system.

Megakaryocyte colony formation was observed in response to recombinantMSF-K130 [SEQ ID NO: 77 and 78] in the murine bone marrow fibrin clotassay. Murine megakaryocytes were identified as acetylcholinesterasepositive cells and a megakaryocyte colony was defined as greater thanthree megakaryocyte cells per unit area in a fibrin clot culture.Recombinant MSF typically stimulated megakaryocyte colonies of three tosix cells/unit area and averaged between 6 to 15 colonies/2.5×10⁵ murinebone marrow cells.

Two types of megakaryocyte colonies were observed in the assay, puremegakaryocyte colonies and megakaryocyte cells with other cell types,termed mixed megakaryocyte colonies. In one fibrin clot, the two colonytypes were at a ratio between 1:1 to 7:3 pure colonies to mixedmegakaryocyte colonies. This ratio was consistent throughout thepurification of recombinant MSF-K130 [SEQ ID NO: 77 and 78]. The numberof megakaryocyte cells/colony and size of megakaryocytes were about thesame for both pure and mixed colonies, some megakaryocytes were smallerin the mixed megakaryocyte colonies.

An increase in bioactivity was usually observed from active MSFfractions obtained from the C4 RP-HPLC column. All three pools from theS Toyopearl cation exchange column gave rise to bioactive MSF protein onRP-HPLC. The final specific activity of MSF after the RP-HPLC step wasgreater than 1×10⁷ units/mg in all three pools. The active peaks werealso positive on the MSF Western Blot.

When RP-HPLC-purified MSF-K130 [SEQ ID NO: 77 and 78] from the A poolwas subjected to SDS-PAGE under non-reducing conditions, bioactiveprotein was extracted from gel slices corresponding to 35-50 kDmolecular weight species. A silver stain gel and Western immunoblot datashowed that 95% of the 35-50 kD recombinant MSF protein reduced to 18-21kD and 5% did not shift upon reduction on a 10-20% acrylamide gradientSDS-PAGE.

The supernatant from COS-1 cells transfected with MSF-K130 cDNA [SEQ IDNO: 77] was variably active on the fibrin clot assay. In each assay thesamples were tested in duplicate and in three dilutions.

B. Human Plasma Clot Megakaryocyte Colony Formation

The human urinary meg-CSF of this invention was also tested for humanactivity on the plasma clot MSF assay described in E. Mazur et al,Blood, 57:277-286 (1981) with modifications. Non-adherent peripheralblood cells were isolated from Leukopacs and frozen in aliquots. Thetest sample was mixed with platelet-poor human AB plasma and 1.25×10⁵cells in 24-well plates and allowed to clot by the addition of calcium.After a 12 day incubation, megakaryocytes were identified using amonoclonal antibody directed against platelet glycoproteins IIb/IIIa anda horseradish peroxidase/anti-peroxidase chromogenic detection system.Recombinant human IL-3 [Genetics Institute, Inc.] was used as a positivecontrol, producing 12-30 megakaryocyte colonies per clot withapproximately 60% pure and 40% mixed megakaryocyte colonies. As in themurine assay, the aplastic dog serum was also used as a positivecontrol, which produced between 5-10 megakaryocyte colonies per clot, ofwhich approximately 50% were pure megakaryocyte colonies containing lessthan 10 cells, and 50% were mixed megakaryocyte colonies containing morethan 40 megakaryocytes. The negative control was Alpha Medium, whichproduced 0-1 megakaryocyte colonies per clot.

The human urinary meg-CSF product from Step 6 of the purification schemeof Example 1 had variable activity in this assay. MSF-K130 [SEQ ID NO:77 and 78] has shown variable activity in the human plasma clotmegakaryocyte colony assay.

C. Synergistic Effects

Recombinant MSF-K130 Cos-1 cell supernatant and purified recombinant MSFwere assayed alone and in combination with other cytokines in thevarious CFU-MEG assay systems, fibrin clot, agar and the human CFU-MEGplasma clot assays.

Variable synergy with IL-3 was observed in the murine bone marrow fibrinclot assay. The stimulation of megakaryocyte colonies increased aboveeither protein alone when both murine IL-3 and MSF-K130 were culturedwith bone marrow cells progenitors in the fibrin clot assay. Asuboptimal level of murine IL-3 (15 units/ml) and an optimal level ofMSF-K130 each stimulate an average of 6-15 CFU-meg/2.5×10⁵ murine bonemarrow cells in the fibrin clot assay. In combination, increasedmegakaryocyte colony stimulation of over 35 megakaryocyte colonies havebeen observed. The ratio of pure megakaryocyte colonies to mixedmegakaryocyte colonies and the size of the megakaryocyte colonies werethe same for the combination cultures as for the individual MSFcultures.

D. E. Coli Expressed MSF Activity

MSF expressed in Escherichia coli as a thioredoxin-MSF-K130 fusionprotein was soluble and active in the fibrin clot assay. E. coliexpressed MSF-K130 stimulated the same range of CFU-meg/2.5×10⁵ murinebone marrow cells as COS derived MSF-K130. This activity was notneutralized by the addition of anti-IL-3 antibody at a level that didneutralize CFU-Meg formation by IL-3. Megakaryocyte colony formationactivity of the MSF-K130 thioredoxin fusion protein from E. coli lysatewas 5×10⁶ dilution units/ml. The specific activity of the MSF-K130thioredoxin fusion protein in E. coli lysate was greater than 1×10⁶U/mg. Thioredoxin was not active in the assay.

EXAMPLE 9 Construction of CHO Cell Lines Expressing High Levels of MSF

One method for producing high levels of the MSF protein of the inventionfrom mammalian cells involves the construction of cells containingmultiple copies of the cDNA encoding the MSF.

The cDNA is co-transfected with an amplifiable marker, e.g., the DHFRgene for which cells containing increasing concentrations ofmethotrexate (MTX) according to the procedures of Kaufman and Sharp, J.Mol. Biol., (1982) supra. This approach can be employed with a number ofdifferent cell types. Alternatively, the MSF cDNA and drug resistanceselection gene (e.g., DHFR) may be introduced into the same vector. Onedesirable vector for this approach is pED4DPC-1. MSF-K130 [SEQ ID NO: 77and 78] and MSF-N141 [SEQ ID NO: 79 and 80] are being expressed invector pEMC3-1, a vector identical to pEO4DPC-1, but in which thepolylinker has been changed (PstI, NotI, SalI, SnaBI, EcoRI, PacI) asdescribed above per pMT21.

For example, the pMT21 vector containing the MSF gene in operativeassociation with other plasmid sequences enabling expression thereof isintroduced into DHFR-deficient CHO cells, DUKX-BII, along with a DHFRexpgression plasmid such as pAdD26SVpA3 [Kaufman, Proc. Natl. Acad. Sci.USA, 82:689-693 (1985)] by calcium phosphate coprecipitation andtransfection. Alternatively, the pED4DPC-1 vector containing the MSFgene in operative association with other plasmid sequences enablingexpression thereof is introduced into DHFR-deficient CHO cells,DUKX-BII, by protoplast fusion or transfection. The MSF gene and DHFRmarker gene are both efficiently expressed when MSF is introduced intopEMC2B1.

DHFR expressing transformants are selected for growth in alpha mediawith dialyzed fetal calf serum. Transformants are checked for expressionof MSF by Westem blotting, bioassay, or RNA blotting and positive poolsare subsequently selected for amplification by growth in increasingconcentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX)as described in Kaufman et al., Mol. Cell Biol., 5:1750 (1983). Theamplified lines are cloned, and MSF protein expression is monitored bythe fibrin clot assay. MSF expression is expected to increase withincreasing levels of MTX resistance.

In any of the expression systems described above, the resulting celllines can be further amplified by appropriate drug selection, resultingcell lines recloned and the level of expression assessed using themurine fibrin clot assay described in Example 4.

The MSF expressing CHO cell lines can be adapted to growth in serum-freemedium. MSF expressed in CHO cells is purified from serum-freeconditioned medium using the same purification scheme as COS-1 cellsupernatant. Homogeneous MSF can be isolated from conditioned mediumfrom the cell line using methods familiar in the art, includingtechniques such as lectin-affinity chromatography, reverse phase HPLC,FPLC and the like.

The foregoing descriptions detail presently preferred embodiments of theinvention. Numerous modifications and variations in practice of thisinvention are expected to occur to those skilled in the art. Suchmodifications and variations are encompassed within the followingclaims.

1. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:28.
 2. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. 